Therapeutic HPV16 vaccines

ABSTRACT

Provided is designer nucleic acid constructs and polypeptides that can be used as therapeutic vaccines against HPV16.

PRIORITY CLAIM

This application claims the benefit under the Paris Convention of thefiling date of European Patent Application Serial No. EP 14 191 660.1,filed Nov. 4, 2014, the contents of the entirety of which areincorporated herein by this reference.

STATEMENT ACCORDING TO 37 C.F.R. §1.821(c) or (e)—SEQUENCE LISTINGSUBMITTED AS A TXT AND PDF FILES

Pursuant to 37 C.F.R. §1.821(c) or (e), files containing a TXT versionand a PDF version of the Sequence Listing have been submittedconcomitant with this application, the contents of which are herebyincorporated by reference.

TECHNICAL FIELD

The disclosure relates to the field of biotechnology and medicine and,more in particular, to nucleic acid constructs and polypeptides that canbe used in therapeutic vaccines against human papillomavirus type 16.

BACKGROUND

The family of human papillomaviruses (HPVs) include more than 100 types(also referred to as subtypes) that are capable of infectingkeratinocytes of the skin or mucosal membranes. Over 40 types of HPV aretypically transmitted through sexual contact and HPV infections of theanogenital region are very common in both men and women. Some sexuallytransmitted HPV types may cause genital warts. Persistent infectionswith “high-risk” HPV types (e.g., types 16, 18, 31, 45)—different fromthe ones that cause skin warts—may progress to precancerous lesions andinvasive cancer, e.g., of the cervix, vulva, vagina, penis, oropharynx,and anus. The majority of HPV infections are spontaneously clearedwithin one to two years after infection. In healthy individualscirculating Th1- and Th2-type CD4+ T-cells specific for the viral earlyproteins E2, E6 and E7 of HPV-16 as well as E6-specific CD8+ T-cells,migrate into the skin upon antigenic challenge, indicating thatsuccessful defense against HPV-16 infection is commonly associated witha systemic effector T-cell response against these viral early antigens.In a minority (˜1%) of infected individuals, HPV infection persists,ultimately resulting in genital neoplastic lesions. Among the high-riskHPVs, HPV16 and HPV18 are the main cause of cervical cancer, togethercausing about 70% of the cases, and these two types also play a majorrole in other HPV-induced cancers such as anal and oropharyngeal cancer.Worldwide, HPV is one of the most important infectious agents causingcancer.

Vaccination against HPV is deemed a feasible strategy to reduce theincidence or effects of infection by HPV (van der Burg and Melief, 2011,Curr. Opinion Immunol. 23:252-257).

Prophylactic HPV vaccines based on virus-like particles (VLPs) formed bythe (envelope) protein L1 of the HPV types 16 and 18, are very efficientin the prevention of persistent infection and the associated disease byHPV16 and HPV18. These vaccines are believed to provide sterile immunityvia the induction of neutralizing antibodies against the L1 proteins.Addition of LlT-based VLPs from additional high-risk HPV types mayfurther increase the breadth of protection conferred by such vaccines.

However, while such vaccines can prevent initial infection (i.e., theyresult in prophylaxis), there is no evidence of a beneficial effect onestablished genital lesions caused by HPV16 and HPV18, so they are notconsidered therapeutic vaccines against HPV (Hildesheim et al., 2007,JAMA 298:743-53).

Despite the introduction of these prophylactic vaccines, large numbersof people have already had or are still at risk of having persistenthigh-risk HPV infections and, therefore, are still at risk of gettingcancer. Therapeutic vaccines for the eradication of established HPVinfections and associated diseases are an urgent unmet medical need.

Some attempts to address this need have been described. For example,clinical trials have been carried out with a variety of differentvaccination strategies, such as a fusion protein consisting of a heatshock protein (Hsp) from Mycobacterium bovis and HPV-16 E7 or consistingof a fusion protein of E6, E7 and L2 from HPV-16 and HPV-18, chimericL1-E7 VLPs, recombinant vaccinia viruses expressing either E6 and E7 ofHPV-16 and HPV-18 or bovine papilloma virus E2, DNA vaccines expressingCTL epitopes of E6 and E7 of HPV-16 and HPV-18, a live-attenuatedListeria monocytogenes (Lm) that secretes the HPV-16 E7 antigen, andsynthetic long-peptides (SLPs) comprising HPV-16 E6 and E7 peptides.While some of these approaches show some, but limited, clinicalefficacy, most have failed, demonstrating that improvement of thecurrent strategies is needed.

Integration of the early HPV proteins E6 and E7 is a necessary step inthe process from infection to cancer and continuous expression of E6 andE7 is required for the maintenance of the neoplastic phenotype ofcervical cancer cells. E6 and E7 are, therefore, considered good targetsfor therapeutic vaccination. As mentioned, some studies have shown thattherapeutic vaccination of women infected with high-risk HPV can induceregression of existing lesions. Kenter et al. showed a durable andcomplete regression in 47% of patients having Vulvar IntraepithelialNeoplasia (VIN) using SLPs derived from the HPV16 E6 and E7 proteins andan adjuvant as a therapeutic vaccine (Kenter et al., 2009, N. Engl. J.Med. 361:1838-47). Similarly, a study in which a protein-based vaccine(TA-CIN, consisting of a fusion protein of HPV16 E6, E7 and L2) wascombined with local immune modulation in VIN 2/3 patients, showedcomplete regression in 63% of patients (Daayana et al., 2010, Br. J.Cancer 102:1129-36). Possible drawbacks of the synthetic long peptidesas a vaccine include manufacturability at large scale and costsassociated therewith, the need for potentially reactogenic adjuvant andthe associated adverse effects associated with immunization (especiallypain and swelling). Due to the high level of discomfort it is not likelythat SLPs will be used in early stage disease when the spontaneousclearance rate is still high. Similarly, due to the need for localimiquimod treatment in the case of TA-CIN treatment, tolerability is asignificant issue as the majority of women experience local and systemicside effects lasting for the duration of imiquimod treatment, which mayaffect daily activities.

A possible alternative is to use nucleic acid based vaccination such asDNA vaccines or viral vaccines encoding the HPV E6 and/or E7 protein forvaccination.

However, the HPV E6 and E7 proteins have oncogenic potential and thusvaccination with vaccines that comprise nucleic acids encoding theseproteins poses a risk of inducing cellular transformation due to thepossibility of prolonged expression of the antigens.

Therefore, in case of genetic vaccination, non-oncogenic/detoxifiedversions of E6 and/or E7 can be used in order to exclude any risk ofcellular transformation due to the vaccination. Loss of oncogenicpotential of wild-type E6 and E7 is commonly achieved by deletion and/orsubstitution of residues known to be important for the function of theseproteins (e.g., Smahel et al., 2001, Virology 281:231-38; Yan et al.,2009, Vaccine 27:431-40; Wieking et al., 2012, Cancer Gene Ther.19:667-74; WO 2009/106362). However, a disadvantage of these approachesis that they carry the risk of removing important T-cell epitopes fromand/or introducing new undesired T-cell epitopes into the proteins, andmay thus not lead to the desired immune response.

In an alternative strategy to remove the oncogenic potential of HPV16 E6and E7, shuffled versions (i.e., polypeptides wherein fragments of thewild-type protein are re-ordered) of the E6 and E7 proteins have beenconstructed (e.g., Öhlschläger et al., 2006, Vaccine 24:2880-93;Oosterhuis et al., 2011, Int. J. Cancer 129:397-406; Oosterhuis et al.,2012, Hum. Gen. Ther. 23:1301-12). However, these approaches would stillrequire manufacturing, formulation and administration of multiplemolecules to ensure inclusion of all possible epitopes of both the E6and E7 proteins, resulting in sub-optimal logistics and relatively highcosts, and moreover the strategies described introduce potentiallystrong non-natural epitopes that are not present in E6 and E7 and sinceimmune responses could be diverted from relevant E6/E7 epitopes towardsuch non-natural epitopes, the described constructs may not have theoptimal immunological characteristics.

Thus, there remains a need in the art for therapeutic vaccines againstHPV, preferably having less of the drawbacks of the approaches describedbefore.

BRIEF SUMMARY

Provided are nucleic acid molecules that encode polypeptides thatcomprise essentially all possible T-cell epitopes of HPV16 oncoproteinsE6 and E7, but nevertheless have a strongly reduced (as compared towild-type (“wt”) E6 and E7), up to non-detectable, transformingactivity, by comprising fragments of the E6 and E7 proteins that havebeen re-ordered, while at the same time containing a minimized number ofundesired neo-epitopes. This is in contrast to molecules previouslypresented by others.

Described is a nucleic acid molecule encoding a polypeptide comprising asequence as set forth in SEQ ID NO:1.

The encoded polypeptide may further comprise a leader sequence.

In certain embodiments, the encoded polypeptide further comprises atleast one epitope of a human papillomavirus (HPV) E2 protein, forexample, an HPV16 E2 protein. The E2 protein may be mutated to decreaseDNA binding, e.g., by a deletion or mutation(s) in its DNA bindingdomain. In certain embodiments, the encoded polypeptide comprises asequence as set forth in SEQ ID NO:3 or SEQ ID NO:5.

In certain embodiments, the nucleic acid sequence is codon-optimized,e.g., for expression in human cells.

In certain embodiments, the nucleic acid molecule comprises apolynucleotide as set forth in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.

Also provided is a vector comprising a nucleic acid molecule asdescribed herein, wherein the molecule encoding the polypeptide isoperably linked to a promoter.

In certain embodiments, the vector is a DNA vector such as a plasmid. Inother embodiments, the vector is a viral vector, such as an MVA vectoror a recombinant adenoviral vector. In certain preferred embodiments,the vector is a recombinant adenovirus.

In certain embodiments, the promoter in the vector is operably coupledto a repressor operator sequence, to which a repressor protein can bindin order to repress expression of the promoter in the presence of therepressor protein. In certain embodiments, the repressor operatorsequence is a TetO sequence or a CuO sequence.

Also provided is a vaccine composition comprising a vector as describedherein, and a pharmaceutically acceptable excipient.

Also provided is a method of inducing an immune response against HPV, inparticular HPV16, in a subject, the method comprising administering tothe subject a vaccine composition as described herein. Also provided isa vaccine as described herein for use in inducing an immune responseagainst HPV, in particular HPV16.

In certain embodiments, the vaccine is administered to the subject morethan once.

Also provided is a method for treating any of: persistent HPV infection(in particular persistent HPV16 infection), vulvar intraepithelialneoplasia (VIN), cervical intraepithelial neoplasia (CIN), vaginalintraepithelial neoplasia (VaIN), anal intraepithelial neoplasia (AIN),cervical cancer (such as cervical squamous cell carcinoma (SCC),oropharyngeal cancer, penile cancer, vaginal cancer, and/or anal cancerin a subject, the method comprising administering to the subject avaccine as described herein. Also provided is a vaccine as describedherein for use in treatment of any of: persistent HPV infection (inparticular persistent HPV16 infection), vulvar intraepithelial neoplasia(VIN), cervical intraepithelial neoplasia (CIN), vaginal intraepithelialneoplasia (VaIN), anal intraepithelial neoplasia (AIN), cervical cancer(such as cervical squamous cell carcinoma (SCC), oropharyngeal cancer,penile cancer, vaginal cancer or anal cancer in a subject.

Also provided is a polypeptide comprising a sequence as set forth in SEQID NO:1, SEQ ID NO:3, or SEQ ID NO:5.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Expression of fusion proteins of HPV16 E6 and E7. HEK-293T cellswere transiently transfected with DNA vectors expressing the transgenesindicated above the figure. Twenty-four hours after transfection thecells were harvested and cell extracts were analyzed by SDS-PAGE andWestern blotting with an antibody against HPV16 E7 (upper panel). Aloading control showing NF-kB (lower panel) confirms similar loading ofcell lysates in all lanes. A molecular weight marker is indicated on theleft. Expected sizes of the fusion proteins: E6E7SH approximately 38kDa; E2E6E7SH and E6E7E2SH approximately 75 kDa, LSE2E6E7SHapproximately 78 kDa.

FIGS. 2A-2C: Colony formation in soft agar. FIG. 2A: Schematicrepresentation of the setup of the soft-agar assay. FIG. 2B:Representative microscopic images at 40× magnification of the cells inagar six weeks post-seeding. The white arrows highlight coloniesobserved in the E7 wt transfected cells. FIG. 2C: Colony quantificationsix weeks post-seeding in agar using the GELCOUNT™ and associatedsoftware. *: p<0.05 (Poisson regression model); **: non-inferior(generalized linear model with non-inferiority margin of 5%).

FIGS. 3A-3D: E6E7SH has lost E6 and E7 activities. FIG. 3A:Representative western blot demonstrating absence of p53 degradation byE6E7SH. Human p53 null NCI-H1299 cells were co-transfected with aplasmid expressing p53 in combination with a plasmid expressing HPV16 E6wild-type, E6E7SH or the empty vector. Non-TF indicates non-transfectedcells. Twenty-four hours after transfection cell lysates were preparedand 30 μg of total protein was loaded on gel. Upper panel—p53 staining,middle panel—E6 staining, lower panel—NF-kB staining (loading control).FIG. 3B: Quantification of p53 levels in four independent assays. Thep53 signal was normalized to the NF-κB signal. FIG. 3C: Western blotdemonstrating lack of pRb degradation by E6E7SH. pRb null Saos-2 cellswere transfected with a plasmid expressing pRb in combination with aplasmid expressing HPV16 E7 wild-type, E6E7SH or the empty vector.Non-TF indicates non-transfected cells. Twenty-four hours aftertransfection cell lysates were prepared and 10 g of total protein wasloaded on gel. Upper panel—pRb staining, middle panel—E7 staining, lowerpanel—NF-κB staining (loading control). FIG. 3D: Quantification of pRblevels in four independent assays. The pRb signal was normalized to theNF-κB signal. *: p<0.05 (ANOVA models); **: non-inferior (testing wasbased on 95% CIs derived from ANOVA models. Non-inferiority margin wasset at 75%).

FIG. 4: E6E7SH does not immortalize primary human epidermalkeratinocytes. Primary human epidermal keratinocytes were transducedwith lentiviruses encoding either the wild-type E6- and E7-encoding openreading frame of HPV16 (E6E7 wt), the E6E7SH sequence or eGFP.Non-transduced donor cells were used as a control. Only expression ofE6E7 wt induces immortalization of primary keratinocytes as indicated bythe extended lifespan and hTERT activation around day 200 (not shown).The cross symbol indicates that the cells died in senescence and couldnot be further cultured. For details see Example 2. Similar results wereobtained in two additional donors (not shown).

FIGS. 5A and 5B: Immune response induced by E6E7SH after DNAimmunization—IFNγ ELISPOT analysis. FIG. 5A: Immunization scheme. CB6F1mice were immunized with DNA plasmids expressing E6E7SH or a plasmidexpressing no transgene (control). Two weeks after immunization the micewere sacrificed and isolated splenocytes were stimulated overnight with15 mer peptide pools corresponding to E7. FIG. 5B: E7-specific immuneresponses in individual mice as measured by IFNγ ELISPOT assays aregiven as spot forming units (SFU) per 10⁶ splenocytes.

FIGS. 6A-6C: Immunogenicity of E6E7SH—IFNγ ELISPOT analysis. FIG. 6A:Immunization scheme. Mice were immunized with adenovectors with insertsas indicated. E7-specific responses at two weeks (FIG. 6B) and at eightweeks (FIG. 6C) were analyzed by IFNγ ELISPOT (represented asspot-forming units (SFU) per 10⁶ splenocytes). The closed circlesrepresent mice immunized with a dosage of 1*10¹⁰ vp, and open circlesrepresent mice immunized with 5*10⁹ vp. The black bar represents thegeometric mean of the responses. The dotted line indicates the lowerdetection limit in the ELISPOT assay. ANOVA Post-hoc Bonferronistatistical analysis was performed on log transformed data. *: p<0.05.For details see Example 3.

FIGS. 7A and 7B: Immunogenicity of E2E6E7SH—E7-tetramer staining. FIG.7A: Immunization scheme. CB6F1 mice were immunized with 1*10¹⁰ vp ofadenovectors expressing the transgenes as indicated. Two weeks afterimmunization the mice were sacrificed and isolated splenocytes analyzedfor the presence of CD8+ cells capable of interacting with E7₄₉₋₅₇-H2-Dbtetramers (FIG. 7B). The percentage of E7-tetramer positive CD8+ T-cellsis indicated on the y-axis. ANOVA Post-hoc Bonferroni statisticalanalysis was performed on log transformed data, the differences betweenthe different E6E7SH variants were not statistically significant.

FIGS. 8A-8C: Immunogenicity of E2E6E7SH—IFNγ ELISPOT analysis. FIG. 8A:Immunization scheme. CB6F1 mice were immunized with adenovectorsexpressing the transgenes indicated below FIGS. 8B and 8C. Two weeksafter immunization the mice were sacrificed and isolated splenocyteswere stimulated overnight with 15mer peptide pools corresponding to E2(FIG. 8B), E6 (not shown) or E7 (FIG. 8C). Responses are given as SFUper 10⁶ splenocytes. ANOVA Post-hoc Bonferroni statistical analysis wasperformed on log transformed data. The E2 response induced byAdenovectors encoding E2 alone is higher than the response induced bythe polypeptides of the disclosure that include the E6 and E7 fragments.The difference is significant for E2 vs E2E6E7SH and E2 vs E6E7E2SH (*:p<0.05). ANOVA Post-hoc Bonferroni statistical analysis was performed onlog transformed data.

FIGS. 9A-9C: Sustained responses in immunized mice. FIG. 9A:Immunization scheme. CB6F1 mice were immunized with 1*10¹⁰ vp of Ad35vectors expressing variants LSE2E6E7SH, E2E6E7SH, E6E7SH, or with anadenovector not expressing a transgene (Empty). Blood samples were takenevery two weeks to determine the percentage E7-specific CD8+ T-cells bytetramer staining. FIG. 9B: Immune responses two weeks afterimmunization. The vector including a leader sequence induced a higherresponse than vectors without the leader sequence; LSE2E6E7SH vsE2E6E7SH (*: p<0.05). FIG. 9C: Kinetics of the responses. ANOVA Post-hocBonferroni statistical analysis was performed on log transformed data ofthe week 2 data set. The E7 response induced by molecules including E2tend to be higher compared to the molecule without E2, though theresults were not statistically significant.

FIGS. 10A and 10B: Use of different Adenoviral vectors to boost immuneresponses. FIG. 10A: Immunization scheme. CB6F1 mice were immunized withan Ad26 vector expressing HPV16 E2E6E7SH (HPV16-Tx) or with an Ad26vector expressing no transgene (empty). Two weeks later theimmunizations were repeated with Ad35-based vectors as indicated belowthe figure. Four weeks after the second immunization the mice weresacrificed and blood samples were used to determine the percentage ofE7-specific CD8+ T-cells by tetramer staining (FIG. 10B). * indicatesthe comparison of Ad26.HPV16-Tx/Ad35.HPV16-Tx versusAd26.HPV16-Tx/Ad35.Empty, p<0.05 (student t-test on log transformeddata, with alpha=0.01 for multiple comparisons).

FIGS. 11A and 11B: Cellular immunogenicity of E2E6E7SH in Rhesusmacaques. FIG. 11A: Immunization scheme. Rhesus macaques were immunizedat day 0: Eight animals received Ad26.HPV16-E2E6E7SH and two controlanimals received Ad26.Empty by intramuscular immunization (i.m.). Aboost immunization was given (Ad26.HPV16-E2E6E7SH or Ad26.Empty) at 8weeks. At 16 weeks, animals received a second boost immunization withAd35 vectors expressing the same E2E6E7SH, while control animalsreceived Ad35.Empty. The dose of adenovectors was 1*10¹¹ vp perimmunization. Blood drawings were performed at several time points. FIG.11B: Cellular immune responses in PBMCs were measured by IFNγ ELISPOT.PBMCs were stimulated with peptide pools corresponding to HPV16 E2, E6or E7 and the number of spot-forming units (SFU) in 1*10⁶ PBMCs aredepicted. The empty control animal (n=2) showed no detectable response.For details see Example 4.

FIGS. 12A-12H: Therapeutic effect of Adenovectors expressingHPV16-2E6E7SH. FIG. 12A: TC-1 injection and immunization scheme CB6F1mice were injected subcutaneously with 1*10⁵ TC-1 cells at day 0. Aftersix days, when tumors were palpable, mice were immunized with two SLPscovering HPV16 E6 and E7 immunodominant epitopes (i.e., HPV16 E6,aa41-65 (KQQLLRREVYDFAFRDLCIVYRDGN; SEQ ID NO:18) and HPV16 E7 aa 43-77(GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR; SEQ ID NO:19)) at 150 μg in afinal volume of 200 μl 0.9% saline supplemented with 5 nmol ODN1826-CpG(FIG. 12B) or Ad26.HPV16-E2E6E7SH (FIG. 12C). Control mice receivedeither CpG alone (FIG. 12D) or Ad26.Empty (FIG. 12E). All mice receiveda boost immunization at day 20. Mice that received Ad26 vectors in theprime immunization were subsequently immunized with the correspondingAd35 vectors. The other mice received, SLP adjuvanted with CpG or CpGalone as in the prime immunizations. FIGS. 12B-12E: Tumor measurement inTC-1 injected mice. Tumor volume was calculated as (width²*length)/2.Mice were sacrificed when tumor volumes surpassed 1000 mm³. Two mice hadto be sacrificed due to weight loss of more than 20% (indicated withasterisks). FIGS. 12F and 12G: Close up of FIGS. 12B and 12C for first35 days. FIG. 12H: Survival after TC-1 injection. The survival of micetreated with Ad.HPV16-E2E6E7SH was significantly increased compared withmice immunized with SLP and CpG (Log-rank test p<0.05). Three miceimmunized with the Ad.HPV16-E2E6E7SH were tumor free at the end of theexperiment (at day 92).

FIGS. 13A and 13B: Adenoviral vectors carrying transgenes encodingeither HPV Ag or LSE2E6E7SH show increased viral yields on cells capableof repressing transgene expression. FIG. 13A: Viral yield assay for Ad35vectors. PER.C6®, PER.C6/CymR, and PER.C6/TetR cells were infected byAd35 vectors carrying GFP-Luc- or HPVAg-encoding transgenes. Thesetransgenes were driven by either CuO- or TetO-containing CMV promoters.Viral yields were determined four days after infection by an Ad35hexon-specific qPCR-based method. FIG. 13B: Viral yield assay for Ad26vectors. PER.C6® and PER.C6/TetR cells were infected by Ad26 vectorscarrying GFP-Luc, HPVAg, or LSE2E6E7SH-encoding transgenes, which wereall driven by a TetO-containing CMV promoter. Viral yields weredetermined three days after infection by an Ad26 hexon-specificqPCR-based method. For details see Example 6.

FIGS. 14A and 14B: Employment of a repressor system to repress transgeneexpression during vector production prevents transgene cassetteinstability in an adenoviral vector carrying an HPVAg-encodingtransgene. An Ad35 vector expressing HPVAg under the control of CMVCuOwas rescued by DNA transfection on either PER.C6® or PER.C6/CymR celllines. Resultant viral plaques were picked—five per cell line—and usedfor consecutive infection rounds on the respective cell lines. FIG. 14A:Analysis of the integrity of the vector transgene cassette region by PCRafter 10 viral passages. PCR products obtained from viral isolatespassaged on PER.C6® and PER.C6/CymR are shown in the middle and rightpanels, respectively. The full-length-appearing PCR products obtainedfor PER.C6®-passaged viral isolates 1, 2, 4, and 5, and those seen forPER.C6/CymR-passaged isolates 1 to 5 were analyzed by Sanger DNAsequencing. Analysis of the chromatogram traces (not shown) revealedthat all isolates grown on PER.C6®, but not those grown on PER.C6/CymR,contained either frameshifting small deletions or premature stopmutations within the coding sequence for HPVAg. FIG. 14B: Analysis ofthe ability of the vectors to express HPVAg after seven viral passages.A549 cells were transduced by the PER.C6®- and PER.C6/CymR-grown viralisolates and HPVAg expression was analyzed by Western Blot using anHPV16 E7-specific antibody. The predicted size for HPVAg is 83 kDa. Fordetails see Example 6.

DETAILED DESCRIPTION

Provided is a nucleic acid molecule encoding a polypeptide comprisingSEQ ID NO:1. The polypeptide is a fusion polypeptide, and is sometimesreferred to herein as the polypeptide of the disclosure, or the fusionpolypeptide of the disclosure. This polypeptide is useful to generate animmune response against the E6 and E7 proteins of HPV16, and thus thenucleic acid molecule can be used as a therapeutic vaccine to preventpersistent HPV16 infection, and diseases associated therewith.

The polypeptide of the disclosure is a carefully designed molecule thatcontains virtually the complete E6 and E7 amino acid sequences of HPV16(it lacks only one amino acid from the C-terminus of the native HPV16 E6protein) in the form of fragments that are re-ordered and partlyoverlapping such that (essentially) all T-cell epitopes of the HPV16 E6and E7 protein are present. Earlier molecules with some potential as HPVvaccines have been described by others (e.g., Kenter et al., 2009, N.Engl. J. Med. 361:1838-47; Daayana et al., 2010, Br. J Cancer102:1129-36; Smahel et al., 2001, Virology 281:231-38; Yan et al., 2009,Vaccine 27:431-40; Öhlschläger et al., 2006, Vaccine 24:2880-93;Oosterhuis et al., 2011, Int. J. Cancer 129:397-406; EP1183368, WO2013/083287), but each of these molecules has one or more drawbacks. Thedesigner polypeptide molecules of the disclosure are advantageous in atleast one and typically several aspects with respect to the approachesdescribed earlier. In particular, advantages of the molecules and/orvectors of the present disclosure include: (i) they have a desiredsafety profile, as the nucleic acid has a strongly reduced (as comparedto native E6 and E7 proteins), up to non-detectable, transformingactivity; (ii) they are single nucleic acid molecules, which are easy tomanufacture at industrial scale in an economically feasible manner, anddo not pose logistic challenges unlike multiple molecule approaches;(iii) the encoded polypeptides comprise essentially all T-cell epitopesof the native HPV16 E6 and E7 proteins; (iv) the design of the encodedpolypeptides has minimized the introduction of undesired potentialstrong neo-epitopes (i.e., epitopes not present in the native E6 and E7proteins); and (v) in certain embodiments, they are not dependent onhighly reactogenic adjuvants to raise a desired immune response. Thus,the molecules hereof represent a major step forward by combining variousadvantageous characteristics in a single design, and are excellentcandidates primarily for therapeutic vaccination against HPV16. Thesemolecules could also possibly work as prophylactic vaccines againstHPV16, meaning that they are likely to prevent persistent infection withHPV16 of vaccinated subjects.

In certain embodiments, by careful design, the number of neo-epitopeswith a length of nine amino acids with a predicted binding affinity<50nM for the 20 most common HLA-A, 20 most common HLA-B and 20 most commonHLA-C alleles was minimized to only one. This is a significantimprovement over constructs described by others, which for a singleshuffled E6 protein already contained more than 30 of such neo-epitopes,and which constructs will highly likely comprise even several moreneo-epitopes in sequences that were appended to these constructs toprevent loss of epitopes (Öhlschläger et al., 2006, Vaccine 24:2880-93).Hence the constructs of the disclosure have a significantly improvedimmunologic profile since chances of an altered immune response ascompared to native E6 and E7 have been minimized in the molecules of thedisclosure, as compared to approaches described by others.

Skilled persons may, using routine techniques, make nucleotidesubstitutions that do not affect the polypeptide sequence encoded by thepolynucleotides described to reflect the codon usage of any particularhost organism in which the polypeptides are to be expressed. Therefore,unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence.Nucleotide sequences that encode proteins and RNA may include introns.

In a preferred embodiment, the nucleic acid molecule encoding thepolypeptide as described herein is codon optimized for expression inmammalian cells, preferably human cells. Methods of codon-optimizationare known and have been described previously (e.g., WO 96/09378, thecontents of which are incorporated herein by this reference). A sequenceis considered codon optimized if at least one non-preferred codon ascompared to a wild type sequence is replaced by a codon that is morepreferred. Herein, a non-preferred codon is a codon that is used lessfrequently in an organism than another codon coding for the same aminoacid, and a codon that is more preferred is a codon that is used morefrequently in an organism than a non-preferred codon. The frequency ofcodon usage for a specific organism can be found in codon frequencytables, such as on the World Wide Web at kazusa.or.jp/codon. Preferablymore than one non-preferred codon, e.g., more than 10%, 40%, 60%, 80% ofnon-preferred codons, preferably most (e.g., at least 90%) or allnon-preferred codons, are replaced by codons that are more preferred.Preferably the most frequently used codons in an organism are used in acodon-optimized sequence. Replacement by preferred codons generallyleads to higher expression.

Nucleic acid sequences can be cloned using routine molecular biologytechniques, or generated de novo by DNA synthesis, which can beperformed using routine procedures by service companies having businessin the field of DNA synthesis and/or molecular cloning (e.g., GENEART®,GENSCRIPT®, INVITROGEN®, EUROFINS®).

It will be appreciated by a skilled person that changes can be made to aprotein, e.g., by amino acid substitutions, deletions, additions, etc.,e.g., using routine molecular biology procedures. Generally,conservative amino acid substitutions may be applied without loss offunction or immunogenicity of a polypeptide. This can be checkedaccording to routine procedures well known to the skilled person.

In certain embodiments, the encoded polypeptide as described hereinfurther comprises a leader sequence, also referred to as signal sequenceor signal peptide. This is a short (typically 5-30 amino acids long)peptide present at the N-terminus of the majority of newly synthesizedproteins that are destined towards the secretory pathway. The presenceof such a sequence may lead to increased expression and immunogenicity.Non-limiting examples that can be used are an IgE leader peptide (see,e.g., U.S. Pat. No. 6,733,994; e.g., having sequence MDWTWILFLVAAATRVHS(SEQ ID NO:7)) or a HAVT20 leader peptide (e.g., having sequenceMACPGFLWALVISTCLEFSMA (SEQ ID NO:9)). One of these can optionally beadded to the N-terminus of a polypeptide of the disclosure. In otherembodiments, a polypeptide as described herein does not comprise aleader sequence.

Diverse types of HPV exist (over 120 types have been identified and arereferred to by number), and generally for each type that needs to becovered by a vaccine, type-specific antigens may need to be incorporatedin the vaccine, although for certain antigens some cross-reactivitymight exist. Types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68,73, and 82 are carcinogenic “high-risk” sexually transmitted HPVs andmay lead to the development of cervical intraepithelial neoplasia (CIN),vulvar intraepithelial neoplasia (VIN), vaginal intraepithelialneoplasia (VaIN), penile intraepithelial neoplasia (PIN), and/or analintraepithelial neoplasia (AIN). The HPV as described herein (i.e., theHPV from which the E6 and E7 fragments in the encoded polypeptide arederived) is HPV16. It can be used for subjects that are infected withHPV16. It may in certain embodiments also suitably be combined withvaccines against other HPV types. In certain embodiments, thiscombination is with a vaccine against HPV of a high risk type asidentified above, e.g., with a vaccine against HPV18. In otherembodiments, the vaccine of the disclosure is combined with a vaccineagainst one or more of HPV-18, -31, -33, -35, -39, -45, -51, -52, -56,-58, -59, -68, -73, or -82. Such combinations could, for instance, beused if the exact type of HPV infection is not yet certain, or if animmune response with a prophylactic effect is desired against more thanone HPV type. Also combinations of the vaccines of the disclosure withvaccines against HPV types that cause genital warts, such as HPV6 and/orHPV11, are envisaged. Sequences of these HPV types and the proteinsencoded thereby (e.g., E6, E7, E2) are available to the skilled personin public databases, such as the GenBank sequence database provided bythe National Center for Technology Information (NCBI).

The polypeptide as described herein comprises SEQ ID NO:1, and in oneembodiment the nucleic acid molecule as described herein comprises SEQID NO:2.

Sequences herein are provided from 5′ to 3′ direction or from N- toC-terminus, as custom in the art.

The polypeptide as described herein comprises the epitopes of HPV16 E6and E7 proteins. In certain embodiments, the polypeptide as describedherein further comprises (and hence the nucleic acid molecule encodingthe polypeptide further encodes) at least one further antigen orepitope(s) of such further antigen. Such a further antigen preferably isan HPV antigen, preferably of the same HPV type as the E6 and E7proteins in the polypeptide, i.e., HPV16. Such a further antigen canthus be an HPV protein or an immunogenic fragment thereof, and incertain embodiments comprises an E2 protein or a fragment thereofcomprising at least one epitope of E2 of HPV, preferably from HPV16.Such further antigens or epitopes could be placed internally between twofragments of E6 and/or E7 in the polypeptide comprising SEQ ID NO:1, butpreferably are fused N-terminally or C-terminally to the E6/E7polypeptide comprising SEQ ID NO:1. Alternatively or in addition, aminoacid sequences can be present that stimulate the immune response. Thus,in certain embodiments provided is nucleic acid molecules as describedherein, encoding a polypeptide comprising SEQ ID NO:1, and wherein thepolypeptide further comprises at least one other antigen, e.g., HPV E2protein or at least one epitope, but preferably more epitopes, thereof.One advantage of the addition of E2 antigen for the instant disclosureis that E2 is known to be expressed early during infection/in low gradelesions where E6 and E7 expression is still very low. During thedevelopment towards cervical cancer E2 expression is lost and as aresult E6 and E7 levels are increased (Yugawa and Kiyono, 2009, Rev.Med. Virol. 19:97-113). Combining epitopes from E2, E6 and E7 in onevaccine allows for treatment in a broad target group of patients,ranging from having persistent infection to invasive cervical cancer (orother HPV16-caused cancers). In certain embodiments, the E2 protein is awild-type E2 protein. In certain other embodiments, the E2 protein has adeletion or one or more mutations in its DNA binding domain (as comparedto a wild type E2 protein). The sequence of the HPV16 E2 protein(NP_041328.1) can be found in the NCBI protein database (on the WorldWide Web at ncbi.nlm.nih.gov/protein) under number NP_041328.1. Severalsingle amino acid changes in E2 such as G293V, K299M, or C300R in theC-terminal part of this protein are known to abrogate DNA binding. Anadvantage of using a variant or fragment of E2 that lacks DNA bindingcapacity is that it could prevent unpredictable transcriptional changesvia direct binding to host cell DNA in the cells where it is expressed.The E2 protein or part or variant thereof can be added internally, butpreferably to the N-terminus or to the C-terminus of the polypeptide ofthe disclosure having SEQ ID NO:1. In one embodiment, the nucleic acidmolecule of the disclosure encodes a polypeptide comprising SEQ ID NO:3.In one embodiment thereof, the nucleic acid molecule of the disclosurecomprises SEQ ID NO:4. In another embodiment, the nucleic acid moleculeof the disclosure encodes a polypeptide comprising SEQ ID NO:5. In oneembodiment thereof, the nucleic acid molecule of the disclosurecomprises SEQ ID NO:6.

It is also possible to make further fusions of the designer polypeptidesof the disclosure with further proteins, e.g., so called carrierproteins, such as Calreticulin, Mycobacterium Tuberculosis heat shockprotein-70, IP10, or Tetanus toxin fragment C (see Oosterhuis et al.,Human Gene Ther., 2012, supra, for more examples), which could furtherenhance the immune response to the HPV E6 and E7 (and optionally E2)epitopes. The disclosure thus also provides such further fusionproteins, and nucleic acids encoding such.

In certain embodiments, a nucleic acid molecule as described herein isincorporated into a vector. A “vector” as used herein, is typically avehicle to artificially carry foreign genetic material into anothercell, where it can be replicated and/or expressed, and as describedherein can be any nucleic acid molecule that incorporates a nucleic acidmolecule as described herein. These can be prepared according to routinemolecular biology techniques such as cloning. Typically such vectors canbe propagated in at least one type of suitable hosts such as bacteria,yeast, insect cells, mammalian cells, and the like. Four major types ofvectors are plasmids, viral vectors, cosmids, and artificialchromosomes. The vector itself is generally a DNA sequence that consistsof an insert (transgene; in the present disclosure the nucleic acidmolecule encoding the fusion polypeptide of the disclosure) and asequence that serves as the “backbone” of the vector. The purpose of avector which transfers genetic information to another cell is typicallyto isolate, multiply, or express the insert in the target cell.Preferably, the sequence encoding the polypeptide is operably linked toa promoter in the vector. The term “operably linked” is intended to meanthat the nucleotide sequence of interest is linked to the promoter in amanner that allows for expression of the nucleotide sequence (e.g., in ahost cell when the vector is introduced into the host cell). Expressionregulatory sequences can be operably linked to a transgene. In certainembodiments, vectors are designed for the expression of the transgene inthe target cell, and generally have a promoter sequence that drivesexpression of the transgene. In certain embodiments, one or more ofroutinely used vector elements such as transcription terminatorsequences, polyadenylation tail sequences, Kozak sequences, UTRs, originof replication, multiple cloning sites, genetic markers, antibioticresistance, and further sequences may be present, and the skilled personcan design a vector such that it has the desired properties, e.g., forreplication in certain cells for propagation and multiplication of thevector, and for expression of the transgene of the vector in targetcells into which the vector is introduced. Vectors comprising thenucleic acid molecule encoding the fusion polypeptide as describedherein, preferably designed for expression in mammalian cells, aresuitable as vaccines as described herein. In certain embodiments, avector as described herein is a plasmid, a cosmid, a yeast artificialchromosome, a bacterial artificial chromosome, a viral vector, or thelike. The person skilled in the art is aware that various promoters canbe used to obtain expression of a gene in host cells. Some well-knownand much used promoters for expression in eukaryotic cells comprisepromoters derived from viruses, such as adenovirus, e.g., the E1Apromoter, promoters derived from cytomegalovirus (CMV), such as the CMVimmediate early (IE) promoter (referred to herein as the CMV promoter)(obtainable, for instance, from pcDNA, INVITROGEN®), promoters derivedfrom Simian Virus 40 (SV40) (e.g., obtainable from pIRES, cat.no.631605, BD Sciences), and the like. Suitable promoters can also bederived from eukaryotic cells, such as metallothionein (MT) promoters,elongation factor 1α (EF-1α) promoter, ubiquitin C or UB6 promoter,actin promoter, an immunoglobulin promoter, heat shock promoters, andthe like (see, e.g., WO 2006/048459). A non-limiting example of asuitable promoter for obtaining expression in eukaryotic cells is aCMV-promoter (U.S. Pat. No. 5,385,839), e.g., the CMV immediate earlypromoter, for instance, comprising nt. −735 to +95 from the CMVimmediate early gene enhancer/promoter, e.g., a CMV promoter as providedherein with a sequence as set forth in SEQ ID NO:13. A polyadenylationsignal, for example, the bovine growth hormone polyA signal (U.S. Pat.No. 5,122,458), may be present behind the transgene(s).

Further regulatory sequences may also be added. The term “regulatorysequence” is used interchangeably with “regulatory element” herein andrefers to a segment of nucleic acid, typically but not limited to DNA,that modulate the transcription of the nucleic acid sequence to which itis operatively linked, and thus acts as a transcriptional modulator. Aregulatory sequence often comprises nucleic acid sequences that aretranscription binding domains that are recognized by the nucleicacid-binding domains of transcriptional proteins and/or transcriptionfactors, enhancers or repressors, etc. For example, it is possible tooperably couple a repressor sequence to the promoter, which repressorsequence can be bound by a repressor protein that can decrease orprevent the expression of the transgene in a production cell line thatexpresses this repressor protein. This may improve genetic stabilityand/or expression levels of the nucleic acid molecule upon passagingand/or when this is produced at high quantities in the production cellline. Such systems have been described in the art. For example, aregulatory sequence could include one or more tetracycline operonoperator sequences (tetO), such that expression is inhibited in thepresence of the tetracycline operon repressor protein (tetR). In theabsence of tetracycline, the tetR protein is able to bind to the tetOsites and repress transcription of a gene operably linked to the tetOsites. In the presence of tetracycline, however, a conformational changein the tetR protein prevents it from binding to the operator sequences,allowing transcription of operably linked genes to occur. In certainembodiments, a nucleic acid molecule, e.g., when present in arecombinant adenovirus vector, of the present disclosure can optionallyinclude tetO operatively linked to a promoter, such that expression ofone or more transgenes is inhibited in recombinant adenoviruses that areproduced in the producer cell line in which tetR protein is expressed.Subsequently, expression would not be inhibited if the recombinantadenovirus is introduced into a subject or into cells that do notexpress the tetR protein (e.g., international patent application WO07/073513). In certain other embodiments, a nucleic acid molecule of thepresent disclosure, e.g., when present in a recombinant adenovirus, canoptionally include a cumate gene-switch system, in which regulation ofexpression is mediated by the binding of the repressor (CymR) to theoperator site (CuO), placed downstream of the promoter (e.g., Mullick etal., BMC Biotechnol. 2006 6:43). As used herein, the term “repressor,”refers to entities (e.g., proteins or other molecules) having thecapacity to inhibit, interfere, retard and/or repress the production ofheterologous protein product of a recombinant expression vector. Forexample, by interfering with a binding site at an appropriate locationalong the expression vector, such as in an expression cassette. Examplesof repressors include tetR, CymR, the lac repressor, the trp repressor,the gal repressor, the lambda repressor, and other appropriaterepressors known in the art. Examples of the use of the tetO/tetRoperator/repressor system and of the CuO/CymR operator/repressor systemare provided herein. Repression of vector transgene expression duringvector propagation can prevent transgene instability, and may increaseyields of vectors having a transgene of the disclosure duringproduction. Hence, in some embodiments, the vectors of the disclosurehave a promoter that can be repressed by binding of a repressor protein,e.g., by having a promoter that is operably coupled to a repressoroperator sequence (e.g., in non-limiting embodiments, a TetO-containingsequence, e.g., the one set forth in SEQ ID NO:11, or a CuO-containingsequence, e.g., the one set forth in SEQ ID NO:12), to which a repressorprotein (e.g., the TetR protein, e.g., having an amino acid sequence asset forth in SEQ ID NO:15, or the CymR protein, e.g., having an aminoacid sequence as set forth in SEQ ID NO:17) can bind.

In certain embodiments, the vector is a plasmid DNA molecule, or afragment thereof. These can be used for DNA vaccination. Other platformsare also possible for use as vectors, for instance, live-attenuateddouble-deleted Listeria monocytogenes strains.

In other embodiments, the vector is a recombinant viral vector, whichmay be replication competent or replication deficient. In certainembodiments, a viral vector comprises a recombinant DNA genome. Incertain embodiments, a vector as described herein is, for instance, arecombinant adenovirus, a recombinant retrovirus, a recombinant poxvirus such as a vaccinia virus (e.g., Modified Vaccinia Ankara (MVA)), arecombinant alphavirus such as Semliki forest virus, a recombinantparamyxovirus, such as a recombinant measles virus, or anotherrecombinant virus. In certain embodiments, a vector as described hereinis an MVA vector.

In preferred embodiments, a vector as described herein is a recombinantadenovirus. Advantages of adenoviruses for use as vaccines include easeof manipulation, good manufacturability at large scale, and an excellentsafety record based on many years of experience in research,development, manufacturing and clinical trials with numerous adenoviralvectors that have been reported. Adenoviral vectors that are used asvaccines generally provide a good immune response to thetransgene-encoded protein, including a cellular immune response. Anadenoviral vector as described herein can be based on any type ofadenovirus, and in certain embodiments is a human adenovirus, which canbe of any serotype. In other embodiments, it is a simian adenovirus,such as chimpanzee or gorilla adenovirus, which can be of any serotype.In certain embodiments, a vector as described herein is of a humanadenovirus serotype 5, 26 or 35. The preparation of recombinantadenoviral vectors is well known in the art. In certain embodiments, anadenoviral vector as described herein is deficient in at least oneessential gene function of the E1 region, e.g., the E1a region and/orthe E1b region, of the adenoviral genome that is required for viralreplication. In certain embodiments, an adenoviral vector as describedherein is deficient in at least part of the non-essential E3 region. Incertain embodiments, the vector is deficient in at least one essentialgene function of the E1 region and at least part of the non-essential E3region.

Adenoviral vectors, methods for construction thereof and methods forpropagating thereof, are well known in the art and are described in, forexample, U.S. Pat. Nos. 5,559,099, 5,837,511, 5,846,782, 5,851,806,5,994,106, 5,994,128, 5,965,541, 5,981,225, 6,040,174, 6,020,191, and6,113,913, and Thomas Shenk, “Adenoviridae and their Replication,” M. S.Horwitz, “Adenoviruses,” Chapters 67 and 68, respectively, in Virology,B. N. Fields et al., eds., 3d ed., Raven Press, Ltd., New York (1996),and other references mentioned herein (each of which is herebyincorporated by this reference in its entirety). Typically, constructionof adenoviral vectors involves the use of standard molecular biologicaltechniques, such as those described in, for example, Sambrook et al.,Molecular Cloning, a Laboratory Manual, 2d ed., Cold Spring HarborPress, Cold Spring Harbor, N.Y. (1989), Watson et al., Recombinant DNA,2d ed., Scientific American Books (1992), and Ausubel et al., CurrentProtocols in Molecular Biology, Wiley Interscience Publishers, NY(1995), and other references mentioned herein.

Particularly preferred serotypes for the recombinant adenovirus arehuman serotype 35 or human serotype 26. Preparation of rAd26 vectors isdescribed, for example, in WO 2007/104792 and in Abbink et al., 2007Virology 81:4654-63. Exemplary genome sequences of Ad26 are found inGenBank Accession EF 153474 and in SEQ ID NO:1 of WO 2007/104792.Preparation of rAd35 vectors is described, for example, in U.S. Pat. No.7,270,811, in WO 00/70071, and in Vogels et al., 2003, J. Virol.77:8263-71. Exemplary genome sequences of Ad35 are found in GenBankAccession AC_000019 and in FIG. 6 of WO 00/70071 (each of which ishereby incorporated by this reference in its entirety).

In certain embodiments, the adenovirus is replication deficient, e.g.,because it contains a deletion in the E1 region of the genome. As knownto the skilled person, in case of deletions of essential regions fromthe adenovirus genome, the functions encoded by these regions have to beprovided in trans, preferably by the producer cell, i.e., when parts orwhole of E1, E2 and/or E4 regions are deleted from the adenovirus, thesehave to be present in the producer cell, for instance, integrated in thegenome thereof, or in the form of so-called helper adenovirus or helperplasmids. The adenovirus may also have a deletion in the E3 region,which is dispensable for replication, and hence such a deletion does nothave to be complemented.

A producer cell (sometimes also referred to in the art and herein as“packaging cell” or “complementing cell”) that can be used can be anyproducer cell wherein a desired adenovirus can be propagated. Forexample the propagation of recombinant adenovirus vectors is done inproducer cells that complement deficiencies in the adenovirus. Suchproducer cells preferably have in their genome at least an adenovirus E1sequence, and thereby are capable of complementing recombinantadenoviruses with a deletion in the E1 region. Any E1-complementingproducer cell can be used, such as human retina cells immortalized byE1, e.g., 911 or PER.C6® cells (see U.S. Pat. No. 5,994,128),E1-transformed amniocytes (see EP Patent 1230354), E1-transformed A549cells (see, e.g., WO 98/39411, U.S. Pat. No. 5,891,690), GH329:HeLa (Gaoet al., 2000, Hum. Gene Ther. 11:213-19), 293, and the like (each ofwhich is hereby incorporated by this reference in its entirety). Incertain embodiments, the producer cells are, for instance, HEK293 cells,or PER.C6® cells, or 911 cells, or IT293SF cells, and the like.Production of adenoviral vectors in producer cells is reviewed in(Kovesdi et al., 2010, Viruses 2:1681-703).

In certain embodiments, an E1-deficient adenovirus comprises the E4-orf6coding sequence of an adenovirus of subgroup C such as Ad5. This allowspropagation of such adenoviruses in well-known complementing cell linesthat express the E1 genes of Ad5, such as, for example, 293 cells orPER.C6® cells (see, e.g., Havenga et al., 2006, J. Gen. Virol.87:2135-43; WO 03/104467, incorporated in its entirety by referenceherein).

“Heterologous nucleic acid” (also referred to herein as “transgene”) invectors of the disclosure is nucleic acid that is not naturally presentin the vector, and according to the present disclosure the nucleic acidmolecule encoding the fusion polypeptide of the disclosure is consideredheterologous nucleic acid when present in a vector. It is introducedinto the vector, for instance, by standard molecular biology techniques.It can, for instance, be cloned into a deleted E1 or E3 region of anadenoviral vector, or in the region between the E4 region and the rITR.A transgene is generally operably linked to expression controlsequences. In preferred embodiments, the transgene is cloned into theE1-region of an adenoviral vector.

Production of vectors such as DNA vectors, or recombinant adenovirusvectors, can be performed according to various methods well known to theperson skilled in the art. Generally, the production entails propagationin cultured cells to generate a substantial amount of vector material,followed by harvest of the vector from the cell culture, and typicallyfollowed by further purification of the vector to remove othersubstances and obtain purified vectors that can be formulated intopharmaceutical compositions (e.g., Hoganson et al., 2002, BioProcessingJ. 1:43-8; Evans et al., 2004, J. Pharm. Sci. 93:2458-75) For example,methods for harvesting adenovirus from cultures of producer cells have,for instance, been extensively described in WO 2005/080556. For example,WO 2010/060719, and WO 2011/098592, both incorporated by referenceherein, describe suitable methods for obtaining and purifying largeamounts of recombinant adenoviruses.

In certain aspects, also provided is a polypeptide that is encoded by anucleic acid molecule as described herein. Such a polypeptide comprisesSEQ ID NO:1. In certain embodiments, such a polypeptide may comprise SEQID NO:3 or SEQ ID NO:5. The characteristics of such a polypeptide are asdescribed above. Such a polypeptide can, for instance, be used directlyas a vaccine against HPV.

The disclosure further provides vaccines comprising nucleic acidmolecules, vectors or polypeptides as described herein, whereinembodiments for each of these aspects can include those as describedabove. In preferred embodiments, a vaccine as described herein comprisesa nucleic acid molecule as described herein. In further preferredembodiments, the vaccine comprises a vector as described herein,preferably a DNA vector, an MVA vector, or a recombinant adenovirusvector.

In certain embodiments, a vaccine as described herein comprises furtheractive ingredients, e.g., nucleic acid molecule encoding at least oneepitope of E6 and/or E7 protein of at least one HPV type different fromHPV16, e.g., a high risk HPV type such as HPV18, -31, -33, -35, -39,-45, -51, -52, -56, -58, -59, -68, -73, or -82.

The term “vaccine” refers to an agent or composition containing anactive component effective to induce a prophylactic and/or therapeuticdegree of immunity in a subject against a certain pathogen or disease,in this case therapeutically against HPV. The vaccine typicallycomprises the nucleic acid molecule, or vector, as described herein, anda pharmaceutically acceptable excipient. Upon administration to asubject, the polypeptide encoded by the nucleic acid molecule asdescribed herein will be expressed in the subject, which will lead to animmune response towards E6 and/or E7 antigenic fragments that arepresent in the polypeptide. The advantage of the instant molecules isthat essentially all T-cell epitopes of HPV16 E6 and E7 are present andthus a T-cell response to any epitope present in wild-type E6 or E7 canbe mounted in the vaccine. Further, the vaccine has all the safety andefficacy advantages as outlined above for the nucleic acid molecules asdescribed herein.

For administering to humans, the disclosure may employ pharmaceuticalcompositions comprising the vector and a pharmaceutically acceptablecarrier or excipient. In the present context, the term “Pharmaceuticallyacceptable” means that the carrier or excipient, at the dosages andconcentrations employed, will not cause any unwanted or harmful effectsin the subjects to which they are administered. Such pharmaceuticallyacceptable excipients are well known in the art (see Remington'sPharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed., MackPublishing Company [1990]; Pharmaceutical Formulation Development ofPeptides and Proteins, S. Frokjaer and L. Hovgaard, eds., Taylor andFrancis [2000]; and Handbook of Pharmaceutical Excipients, 3rd edition,A. Kibbe, Ed., Pharmaceutical Press [2000]). An excipient is generally apharmacologically inactive substance formulated with the activeingredient of a medication. Excipients are commonly used to bulk upformulations that contain potent active ingredients (thus often referredto as “bulking agents,” “fillers,” or “diluents”), to allow convenientand accurate dispensation of a drug substance when producing a dosageform. They also can serve various therapeutic-enhancing purposes, suchas facilitating drug absorption or solubility, or other pharmacokineticconsiderations. Excipients can also be useful in the manufacturingprocess, to aid in the handling of the active substance concerned suchas by facilitating powder flowability or non-stick properties, inaddition to aiding in vitro stability such as prevention of denaturationover the expected shelf life. The selection of appropriate excipientsalso depends upon the route of administration and the dosage form, aswell as the active ingredient and other factors.

The purified nucleic acid molecule, vector or polypeptide preferably isformulated and administered as a sterile solution although it is alsopossible to utilize lyophilized preparations. Sterile solutions areprepared by sterile filtration or by other methods known per se in theart. The solutions are then lyophilized or filled into pharmaceuticaldosage containers. The pH of the solution generally is in the range ofpH 3.0 to 9.5, e.g., pH 5.0 to 7.5. The nucleic acid molecule or vectoror polypeptide typically is in a solution having a suitable buffer, andthe solution of vector may also contain a salt. Optionally stabilizingagent may be present, such as albumin. In certain embodiments, detergentis added. In certain embodiments, vaccine may be formulated into aninjectable preparation. These formulations contain effective amounts ofnucleic acid molecule, vector or polypeptide are either sterile liquidsolutions, liquid suspensions or lyophilized versions and optionallycontain stabilizers or excipients.

For instance, recombinant adenovirus vector may be stored in the bufferthat is also used for the Adenovirus World Standard (Hoganson et al.,2002, Bioprocessing J. 1:43-8): 20 mM Tris pH 8, 25 mM NaCl, 2.5%glycerol. Another useful formulation buffer suitable for administrationto humans is 20 mM Tris, 2 mM MgCl₂, 25 mM NaCl, sucrose 10% w/v,polysorbate-80 0.02% w/v. Another formulation buffer that is suitablefor recombinant adenovirus comprises 10-25 mM citrate buffer pH 5.9-6.2,4-6% (w/w) hydroxypropyl-beta-cyclodextrin (HBCD), 70-100 mM NaCl,0.018-0.035% (w/w) polysorbate-80, and optionally 0.3-0.45% (w/w)ethanol. Obviously, many other buffers can be used, and several examplesof suitable formulations for the storage and for pharmaceuticaladministration of purified vectors are known.

In certain embodiments, a composition comprising the vector furthercomprises one or more adjuvants. Adjuvants are known in the art tofurther increase the immune response to an applied antigenicdeterminant. The terms “adjuvant” and “immune stimulant” are usedinterchangeably herein, and are defined as one or more substances thatcause stimulation of the immune system. In this context, an adjuvant isused to enhance an immune response to the polypeptides encoded by thenucleic acid molecules in the vectors of the disclosure. Examples ofsuitable adjuvants include aluminum salts such as aluminum hydroxideand/or aluminum phosphate and/or aluminum potassium phosphate;oil-emulsion compositions (or oil-in-water compositions), includingsqualene-water emulsions, such as MF59 (see, e.g., WO 90/14837); saponinformulations, such as, for example, QS21 and Immunostimulating Complexes(ISCOMS) (see, e.g., U.S. Pat. No. 5,057,540; WO 90/03184, WO 96/11711,WO 2004/004762, WO 2005/002620); bacterial or microbial derivatives,examples of which are monophosphoryl lipid A (MPL), 3-O-deacylated MPL(3dMPL), CpG-motif containing oligonucleotides, ADP-ribosylatingbacterial toxins or mutants thereof, such as E. coli heat labileenterotoxin LT, cholera toxin CT, and the like. It is also possible touse vector-encoded adjuvant, e.g., by using heterologous nucleic acidthat encodes a fusion of the oligomerization domain of C4-bindingprotein (C4 bp) to the antigen of interest (e.g., Solabomi et al., 2008,Infect. Immun. 76:3817-23), or by using a vector encoding both thetransgene of interest and a TLR-3 agonist such as heterologous dsRNA(e.g., WO 2007/100908), or the like.

In other embodiments, the compositions of the disclosure do not compriseadjuvants.

Pharmaceutical compositions may be administered to a subject, e.g., ahuman subject. The total dose of the vaccine active component providedto a subject during one administration can be varied as is known to theskilled practitioner, and for adenovirus is generally between 1×10⁷viral particles (vp) and 1×10¹² vp, preferably between 1×10⁸ vp and1×10¹¹ vp, for instance, between 3×10⁸ and 5×10¹⁰ vp, for instance,between 10⁹ and 3×10¹⁰ vp. For a DNA vaccine, total amounts of DNA peradministration may, for instance, be between 1 μg and 10 mg. If a genegun is used for administration, typically low amounts are used, e.g., 10μg. For intramuscular injection, typically higher amounts are used,e.g., up to 5 mg.

Administration of pharmaceutical compositions can be performed usingstandard routes of administration. Non-limiting embodiments includeparenteral administration, such as by injection, e.g., intradermal,intramuscular, etc., or subcutaneous or transcutaneous, or mucosaladministration, e.g., intranasal, oral, intravaginal, rectal, and thelike. In one embodiment a composition is administered by intramuscularinjection, e.g., into the deltoid muscle of the arm, or vastus lateralismuscle of the thigh. In certain embodiments the vaccine is a DNAvaccine, and this can, for instance, be administered intradermally,e.g., by DNA tattooing (see, e.g., Oosterhuis et al., 2012, Curr. TopMicrobiol. Immunol. 351:221-50); this route is also feasible foradenoviral vectors. In certain embodiments a composition as describedherein comprises an adenoviral vector and is administered byintramuscular injection. The skilled person knows the variouspossibilities to administer a composition, such as a vaccine in order toinduce an immune response to the antigen(s) in the vaccine.

A subject as used herein preferably is a mammal, for instance, a rodent,e.g., a mouse, or a non-human-primate, or a human. Preferably, thesubject is a human subject.

The vaccines of the disclosure can be used to treat patients having oneof various stages of diseases caused by HPV (in particular type 16),from incident and persistent HPV infection as such (e.g., as detected byHPV DNA testing), thus before (pre-)cancerous lesions are formed, aswell as cervical intraepithelial neoplasia (CIN; also known as cervicaldysplasia and cervical interstitial neoplasia, which is the potentiallypremalignant transformation and abnormal growth (dysplasia) of squamouscells on the surface of the cervix) up to and including cervical cancer(such as cervical squamous cell carcinoma (SCC). In addition, otherHPV-induced neoplasias, such as vulvar intraepithelial neoplasia (VIN),vaginal intraepithelial neoplasia (VaIN), penile intraepithelialneoplasia (PIN), anal intraepithelial neoplasia (AIN) can be targeted aswell as more advanced stages of oropharyngeal cancer (also known ashead- and neck cancer), penile cancer, vaginal cancer, vulvar cancer andanal cancer. The vaccines of the disclosure thus can target a wide rangeof HPV induced lesions, and are likely most effective at theprecancerous stages of HPV-induced disease, e.g., at the (persistent)infection and/or the neoplasia stages, where expression of E2, E6 and/orE7 is highest. It is also possible to combine the treatment using avaccine of the disclosure with compounds that counteract or can overcomeimmune escape mechanisms in advanced cancer cells e.g., anti-PD1/PD-L1antibodies, anti-CTLA-4 antibodies such as Ipilimumab, anti-LAG-3antibodies, anti-CD25 antibodies, IDO-inhibitors, CD40 agonisticantibodies, CD137 agonistic antibodies, etc. (see, e.g., Hamid andCarvajal, 2013, Expert Opinion Biol. Ther. 13:847-861; Mellman et al.,2011, Nature Rev. 480:480-89). The therapeutic vaccination method couldin principle also be used for treating external genital warts orprecursors thereof in case the vaccine comprises further (sequencesencoding) E6 and/or E7 of an HPV type causing external genital warts andis administered to a subject infected by such an HPV type.

As used herein, “treating” means administration of the vaccine to inducea therapeutic immune response against cells that express (epitopes of)HPV16 E6 and/or E7 in the patient, which leads to at least reduction ofthe level of and preferably complete removal of HPV16 infection, whichresults in at least slowing and preferably stopping the progress ofHPV16-caused disease such as neoplasias and/or symptoms thereof.Preferably treatment with the vaccine results also in remission of moreadvanced stages of HPV-induced cancers. It is preferred to administerthe vaccine to patients that have an established HPV infection that hasbeen typed, so that the vaccine that encodes the polypeptide of thecorresponding HPV type can be administered. In the absence of screeningthe vaccine can also be administered in the part of the population thatis likely to be HPV infected, i.e., sexually active people. It is alsopossible to administer a vaccine of the disclosure to subjects that havenot been infected by HPV16, e.g., for prophylactic use, possibly incombination with a vaccine against another HPV type by which the patienthas been infected, or alternatively in non-infected subjects. A vaccineof the disclosure can also be administered to a subject that is subjectto further treatment by other means, e.g., surgery (removal of a lesioncaused by HPV16 infection), or treatment with imiquimod (comprising aTLR-7/8 agonist, see, e.g., Dayaana et al., 2010, Br. J. Cancer102:1129-36). The effect of the treatment can be measured either bycytology or by HPV testing.

The vaccination comprises administering the vaccine of the disclosure toa subject or patient at least once. It is also possible to provide oneor more booster administrations of one or more further vaccines. If aboosting vaccination is performed, typically, such a boostingvaccination will be administered to the same subject at a moment betweenone week and one year, preferably between two weeks and four months,after administering an immunogenic composition with the same antigen tothe subject for the first time (which is in such cases referred to as“priming vaccination”). In alternative boosting regimens, it is alsopossible to administer different vectors, e.g., one or more adenovirusesof different serotype, or other vectors such as MVA, or DNA, or protein,to the subject as a priming or boosting vaccination. In certainembodiments, the same form of a vaccine of the disclosure isadministered at least twice to the same patient in a prime-boostregimen, e.g., with the same recombinant adenovirus (such as Ad26) asdescribed herein. In certain embodiments, a vaccine of the disclosure isadministered at least twice in a prime-boost regimen, but the vector ofthe vaccine is different, e.g., two different serotypes of adenoviralvectors are used, e.g., priming with recombinant Ad26 and boosting withrecombinant Ad35, or vice versa; or priming with DNA and boosting withan adenoviral vector, or vice versa; or priming with an adenoviralvector and boosting with an MVA vector, or vice versa. In certainembodiments, a vaccine as described herein is administered at leastthree times, in a prime-boost-boost regimen. Further boosteradministrations might be added to the regimen.

It is also an aspect of the disclosure to induce a CTL response againstHPV16 in a subject, comprising administering a vaccine as describedherein to the subject.

Provided is also the following non-limiting embodiments:

1) a nucleic acid molecule encoding a polypeptide comprising SEQ IDNO:1;

2) a nucleic acid molecule according to embodiment 1, wherein thepolypeptide further comprises at least part of HPV E2 protein;

3) a nucleic acid molecule according to embodiment 2, wherein the atleast part of the HPV E2 protein is from the E2 protein of HPV16;

4) a nucleic acid molecule according to embodiment 2, wherein thepolypeptide comprises at least part of the E2 protein fused to theN-terminal side of the polypeptide with SEQ ID NO:1;

5) a nucleic acid molecule according to embodiment 2, wherein thepolypeptide comprises at least part of the E2 protein fused to theC-terminal side of the polypeptide with SEQ ID NO: 1;

6) a nucleic acid molecule according to embodiment 3, wherein thepolypeptide comprises at least part of the E2 protein fused to theN-terminal side of the polypeptide with SEQ ID NO:1;

7) a nucleic acid molecule according to embodiment 3, wherein thepolypeptide comprises at least part of the E2 protein fused to theC-terminal side of the polypeptide with SEQ ID NO: 1;

8) a nucleic acid molecule according to embodiment 2, wherein the atleast part of the E2 protein comprises a variant of the E2 protein witha mutation that abrogates DNA binding of E2;

9) a nucleic acid molecule according to embodiment 3, wherein the atleast part of the E2 protein comprises a variant of the E2 protein witha mutation that abrogates DNA binding of E2;

10) a nucleic acid molecule according to embodiment 4, wherein the atleast part of the E2 protein comprises a variant of the E2 protein witha mutation that abrogates DNA binding of E2;

11) a nucleic acid molecule according to embodiment 5, wherein the atleast part of the E2 protein comprises a variant of the E2 protein witha mutation that abrogates DNA binding of E2;

12) a nucleic acid molecule according to embodiment 6, wherein the atleast part of the E2 protein comprises a variant of the E2 protein witha mutation that abrogates DNA binding of E2;

13) a nucleic acid molecule according to embodiment 7, wherein the atleast part of the E2 protein comprises a variant of the E2 protein witha mutation that abrogates DNA binding of E2;

14) a vector comprising a nucleic acid molecule according to embodiment1, wherein a sequence encoding the polypeptide is operably linked to apromoter;

15) a vector comprising a nucleic acid molecule according to embodiment2, wherein a sequence encoding the polypeptide is operably linked to apromoter;

16) a vector comprising a nucleic acid molecule according to embodiment3, wherein a sequence encoding the polypeptide is operably linked to apromoter;

17) a vector comprising a nucleic acid molecule according to embodiment4, wherein a sequence encoding the polypeptide is operably linked to apromoter;

18) a vector comprising a nucleic acid molecule according to embodiment5, wherein a sequence encoding the polypeptide is operably linked to apromoter;

19) a vector comprising a nucleic acid molecule according to embodiment6, wherein a sequence encoding the polypeptide is operably linked to apromoter;

20) a vector comprising a nucleic acid molecule according to embodiment7, wherein a sequence encoding the polypeptide is operably linked to apromoter;

21) a vector comprising a nucleic acid molecule according to embodiment8, wherein a sequence encoding the polypeptide is operably linked to apromoter;

22) a vector comprising a nucleic acid molecule according to embodiment9, wherein a sequence encoding the polypeptide is operably linked to apromoter;

23) a vector comprising a nucleic acid molecule according to embodiment10, wherein a sequence encoding the polypeptide is operably linked to apromoter;

24) a vector comprising a nucleic acid molecule according to embodiment11, wherein a sequence encoding the polypeptide is operably linked to apromoter;

25) a vector comprising a nucleic acid molecule according to embodiment12, wherein a sequence encoding the polypeptide is operably linked to apromoter;

26) a vector comprising a nucleic acid molecule according to embodiment13, wherein a sequence encoding the polypeptide is operably linked to apromoter;

27) a vector according to embodiment 14, wherein the vector is anadenovirus;

28) a vector according to embodiment 15, wherein the vector is anadenovirus;

29) a vector according to embodiment 16, wherein the vector is anadenovirus;

30) a vector according to embodiment 17, wherein the vector is anadenovirus;

31) a vector according to embodiment 18, wherein the vector is anadenovirus;

32) a vector according to embodiment 19, wherein the vector is anadenovirus;

33) a vector according to embodiment 20, wherein the vector is anadenovirus;

34) a vector according to embodiment 21, wherein the vector is anadenovirus;

35) a vector according to embodiment 22, wherein the vector is anadenovirus;

36) a vector according to embodiment 23, wherein the vector is anadenovirus;

37) a vector according to embodiment 24, wherein the vector is anadenovirus;

38) a vector according to embodiment 25, wherein the vector is anadenovirus;

39) a vector according to embodiment 26, wherein the vector is anadenovirus;

40) a vector according to embodiment 27, wherein the adenovirus is ahuman adenovirus of serotype 26;

41) a vector according to embodiment 28, wherein the adenovirus is ahuman adenovirus of serotype 26;

42) a vector according to embodiment 29, wherein the adenovirus is ahuman adenovirus of serotype 26;

43) a vector according to embodiment 30, wherein the adenovirus is ahuman adenovirus of serotype 26;

44) a vector according to embodiment 31, wherein the adenovirus is ahuman adenovirus of serotype 26;

45) a vector according to embodiment 32, wherein the adenovirus is ahuman adenovirus of serotype 26;

46) a vector according to embodiment 33, wherein the adenovirus is ahuman adenovirus of serotype 26;

47) a vector according to embodiment 34, wherein the adenovirus is ahuman adenovirus of serotype 26;

48) a vector according to embodiment 35, wherein the adenovirus is ahuman adenovirus of serotype 26;

49) a vector according to embodiment 36, wherein the adenovirus is ahuman adenovirus of serotype 26;

50) a vector according to embodiment 37, wherein the adenovirus is ahuman adenovirus of serotype 26;

51) a vector according to embodiment 38, wherein the adenovirus is ahuman adenovirus of serotype 26;

52) a vector according to embodiment 39, wherein the adenovirus is ahuman adenovirus of serotype 26;

53) a vector according to embodiment 28, wherein the adenovirus is ahuman adenovirus of serotype 35;

54) a vector according to embodiment 29, wherein the adenovirus is ahuman adenovirus of serotype 35;

55) a vector according to embodiment 30, wherein the adenovirus is ahuman adenovirus of serotype 35;

56) a vector according to embodiment 31, wherein the adenovirus is ahuman adenovirus of serotype 35;

57) a vector according to embodiment 32, wherein the adenovirus is ahuman adenovirus of serotype 35;

58) a vector according to embodiment 33, wherein the adenovirus is ahuman adenovirus of serotype 35;

59) a vector according to embodiment 34, wherein the adenovirus is ahuman adenovirus of serotype 35;

60) a vector according to embodiment 35, wherein the adenovirus is ahuman adenovirus of serotype 35;

61) a vector according to embodiment 36, wherein the adenovirus is ahuman adenovirus of serotype 35;

62) a vector according to embodiment 37, wherein the adenovirus is ahuman adenovirus of serotype 35;

63) a vector according to embodiment 38, wherein the adenovirus is ahuman adenovirus of serotype 35;

64) a vector according to embodiment 39, wherein the adenovirus is ahuman adenovirus of serotype 35;

65) a vaccine composition comprising a vector according to embodiment14, and a pharmaceutically acceptable excipient;

66) a vaccine composition comprising a vector according to embodiment15, and a pharmaceutically acceptable excipient;

67) a vaccine composition comprising a vector according to embodiment16, and a pharmaceutically acceptable excipient;

68) a vaccine composition comprising a vector according to embodiment17, and a pharmaceutically acceptable excipient;

69) a vaccine composition comprising a vector according to embodiment18, and a pharmaceutically acceptable excipient;

70) a vaccine composition comprising a vector according to embodiment19, and a pharmaceutically acceptable excipient;

71) a vaccine composition comprising a vector according to embodiment20, and a pharmaceutically acceptable excipient;

72) a vaccine composition comprising a vector according to embodiment21, and a pharmaceutically acceptable excipient;

73) a vaccine composition comprising a vector according to embodiment22, and a pharmaceutically acceptable excipient;

74) a vaccine composition comprising a vector according to embodiment23, and a pharmaceutically acceptable excipient;

75) a vaccine composition comprising a vector according to embodiment24, and a pharmaceutically acceptable excipient;

76) a vaccine composition comprising a vector according to embodiment25, and a pharmaceutically acceptable excipient;

77) a vaccine composition comprising a vector according to embodiment26, and a pharmaceutically acceptable excipient;

78) a vaccine composition comprising a vector according to embodiment27, and a pharmaceutically acceptable excipient;

79) a vaccine composition comprising a vector according to embodiment28, and a pharmaceutically acceptable excipient;

80) a vaccine composition comprising a vector according to embodiment29, and a pharmaceutically acceptable excipient;

81) a vaccine composition comprising a vector according to embodiment30, and a pharmaceutically acceptable excipient;

82) a vaccine composition comprising a vector according to embodiment31, and a pharmaceutically acceptable excipient;

83) a vaccine composition comprising a vector according to embodiment32, and a pharmaceutically acceptable excipient;

84) a vaccine composition comprising a vector according to embodiment33, and a pharmaceutically acceptable excipient;

85) a vaccine composition comprising a vector according to embodiment34, and a pharmaceutically acceptable excipient;

86) a vaccine composition comprising a vector according to embodiment35, and a pharmaceutically acceptable excipient;

87) a vaccine composition comprising a vector according to embodiment36, and a pharmaceutically acceptable excipient;

88) a vaccine composition comprising a vector according to embodiment37, and a pharmaceutically acceptable excipient;

89) a vaccine composition comprising a vector according to embodiment38, and a pharmaceutically acceptable excipient;

90) a vaccine composition comprising a vector according to embodiment39, and a pharmaceutically acceptable excipient;

91) a vaccine composition comprising a vector according to embodiment40, and a pharmaceutically acceptable excipient;

92) a vaccine composition comprising a vector according to embodiment41, and a pharmaceutically acceptable excipient;

93) a vaccine composition comprising a vector according to embodiment42, and a pharmaceutically acceptable excipient;

94) a vaccine composition comprising a vector according to embodiment43, and a pharmaceutically acceptable excipient;

95) a vaccine composition comprising a vector according to embodiment44, and a pharmaceutically acceptable excipient;

96) a vaccine composition comprising a vector according to embodiment45, and a pharmaceutically acceptable excipient;

97) a vaccine composition comprising a vector according to embodiment46, and a pharmaceutically acceptable excipient;

98) a vaccine composition comprising a vector according to embodiment47, and a pharmaceutically acceptable excipient;

99) a vaccine composition comprising a vector according to embodiment48, and a pharmaceutically acceptable excipient;

100) a vaccine composition comprising a vector according to embodiment49, and a pharmaceutically acceptable excipient;

101) a vaccine composition comprising a vector according to embodiment50, and a pharmaceutically acceptable excipient;

102) a vaccine composition comprising a vector according to embodiment51, and a pharmaceutically acceptable excipient;

103) a vaccine composition comprising a vector according to embodiment52, and a pharmaceutically acceptable excipient;

104) a vaccine composition comprising a vector according to embodiment53, and a pharmaceutically acceptable excipient;

105) a vaccine composition comprising a vector according to embodiment54, and a pharmaceutically acceptable excipient;

106) a vaccine composition comprising a vector according to embodiment55, and a pharmaceutically acceptable excipient;

107) a vaccine composition comprising a vector according to embodiment56, and a pharmaceutically acceptable excipient;

108) a vaccine composition comprising a vector according to embodiment57, and a pharmaceutically acceptable excipient;

109) a vaccine composition comprising a vector according to embodiment58, and a pharmaceutically acceptable excipient;

110) a vaccine composition comprising a vector according to embodiment59, and a pharmaceutically acceptable excipient;

111) a vaccine composition comprising a vector according to embodiment60, and a pharmaceutically acceptable excipient;

112) a vaccine composition comprising a vector according to embodiment61, and a pharmaceutically acceptable excipient;

113) a vaccine composition comprising a vector according to embodiment62, and a pharmaceutically acceptable excipient;

114) a vaccine composition comprising a vector according to embodiment63, and a pharmaceutically acceptable excipient;

115) a vaccine composition comprising a vector according to embodiment64, and a pharmaceutically acceptable excipient;

116) a method for inducing an immune response against HPV in a subject,comprising administering to the subject a vaccine composition accordingto any one of embodiments 65-115;

117) a method for treating persistent HPV (type 16) infection,comprising administering a vaccine according to any one of embodiments65-115 to a subject that suffers from persistent HPV infection;

118) a method for treating vulvar intraepithelial neoplasia (VIN) (withunderlying HPV type 16 infection), the method comprising administering avaccine according to any one of embodiments 65-115 to a subject thatsuffers from VIN;

119) a method for treating vulvar cancer (with underlying HPV type 16infection), the method comprising administering a vaccine according toany one of embodiments 65-115 to a subject that suffers from vulvarcancer;

120) a method for treating cervical intraepithelial neoplasia (CIN)(with underlying HPV type 16 infection), the method comprisingadministering a vaccine according to any one of embodiments 65-115 to asubject that suffers from CIN;

121) a method for treating cervical cancer (with underlying HPV type 16infection), the method comprising administering a vaccine according toany one of embodiments 65-115 to a subject that suffers from cervicalcancer;

122) a method for treating oropharyngeal cancer (with underlying HPVtype 16 infection), the method comprising administering a vaccineaccording to any one of embodiments 65-115 to a subject that suffersfrom oropharyngeal cancer;

123) a method for treating penile intraepithelial neoplasia (PIN) (withunderlying HPV type 16 infection), the method comprising administering avaccine according to any one of embodiments 65-115 to a subject thatsuffers from PIN;

124) a method for treating penile cancer (with underlying HPV type 16infection), the method comprising administering a vaccine according toany one of embodiments 65-115 to a subject that suffers from penilecancer;

125) a method for treating vaginal intraepithelial neoplasia (VaIN)(with underlying HPV type 16 infection), the method comprisingadministering a vaccine according to any one of embodiments 65-115 to asubject that suffers from VaIN;

126) a method for treating vaginal cancer (with underlying HPV type 16infection), the method comprising administering a vaccine according toany one of embodiments 65-115 to a subject that suffers from vaginalcancer;

127) a method for treating anal intraepithelial neoplasia (AIN) (withunderlying HPV type 16 infection), the method comprising administering avaccine according to any one of embodiments 65-115 to a subject thatsuffers from AIN;

128) a method for treating anal cancer (with underlying HPV type 16infection), the method comprising administering a vaccine according toany one of embodiments 65-115 to a subject that suffers from analcancer;

129) a polypeptide comprising SEQ ID NO:1;

130) a polypeptide according to embodiment 129, wherein the polypeptidefurther comprises at least part of HPV E2 protein;

131) a polypeptide according to embodiment 130, wherein the at leastpart of the HPV E2 protein is from the E2 protein of HPV16;

132) a polypeptide according to embodiment 130, wherein at least part ofthe E2 protein is fused to the N-terminal side of the polypeptide withSEQ ID NO:1;

133) a polypeptide according to embodiment 130, wherein at least part ofthe E2 protein is fused to the C-terminal side of the polypeptide withSEQ ID NO: 1;

134) a polypeptide according to embodiment 131, wherein at least part ofthe E2 protein is fused to the N-terminal side of the polypeptide withSEQ ID NO:1;

135) a polypeptide according to embodiment 131, wherein at least part ofthe E2 protein is fused to the C-terminal side of the polypeptide withSEQ ID NO:1;

136) a polypeptide according to embodiment 130, wherein the at leastpart of the E2 protein comprises a variant of the E2 protein with amutation that abrogates DNA binding of E2;

137) a polypeptide according to embodiment 131, wherein the at leastpart of the E2 protein comprises a variant of the E2 protein with amutation that abrogates DNA binding of E2;

138) a polypeptide according to embodiment 132, wherein the at leastpart of the E2 protein comprises a variant of the E2 protein with amutation that abrogates DNA binding of E2;

139) a polypeptide according to embodiment 133, wherein the at leastpart of the E2 protein comprises a variant of the E2 protein with amutation that abrogates DNA binding of E2;

140) a polypeptide according to embodiment 134, wherein the at leastpart of the E2 protein comprises a variant of the E2 protein with amutation that abrogates DNA binding of E2;

141) a polypeptide according to embodiment 135, wherein the at leastpart of the E2 protein comprises a variant of the E2 protein with amutation that abrogates DNA binding of E2;

142) a nucleic acid molecule according to embodiment 3, encoding apolypeptide according to SEQ ID NO:3;

143) a nucleic acid molecule according to embodiment 3, encoding apolypeptide according to SEQ ID NO:5;

144) a vector encoding a nucleic acid molecule according to embodiment142, wherein a sequence encoding the polypeptide is operably linked to apromoter;

145) a vector encoding a nucleic acid molecule according to embodiment143, wherein a sequence encoding the polypeptide is operably linked to apromoter;

146) a vector according to embodiment 144, wherein the vector is anadenovirus;

147) a vector according to embodiment 145, wherein the vector is anadenovirus;

148) a vector according to embodiment 146, wherein the adenovirus is ahuman adenovirus of serotype 26;

149) a vector according to embodiment 147, wherein the adenovirus is ahuman adenovirus of serotype 26;

150) a vector according to embodiment 146, wherein the adenovirus is ahuman adenovirus of serotype 35;

151) a vector according to embodiment 147, wherein the adenovirus is ahuman adenovirus of serotype 35;

152) a vaccine composition comprising a vector according to embodiment144, and a pharmaceutically acceptable excipient;

153) a vaccine composition comprising a vector according to embodiment145, and a pharmaceutically acceptable excipient;

154) a vaccine composition comprising a vector according to embodiment146, and a pharmaceutically acceptable excipient;

155) a vaccine composition comprising a vector according to embodiment147, and a pharmaceutically acceptable excipient;

156) a vaccine composition comprising a vector according to embodiment148, and a pharmaceutically acceptable excipient;

157) a vaccine composition comprising a vector according to embodiment149, and a pharmaceutically acceptable excipient;

158) a vaccine composition comprising a vector according to embodiment150, and a pharmaceutically acceptable excipient;

159) a vaccine composition comprising a vector according to embodiment151, and a pharmaceutically acceptable excipient;

160) a method for inducing an immune response against HPV in a subject,the method comprising administering to the subject a vaccine compositionaccording to any one of embodiments 152-159;

161) a method for treating vulvar intraepithelial neoplasia (VIN), themethod comprising administering a vaccine according to any one ofembodiments 152-159 to a subject that suffers from VIN;

162) a method for treating vulvar cancer, the method comprisingadministering a vaccine according to any one of embodiments 152-159 to asubject that suffers from vulvar cancer;

163) a method for treating cervical intraepithelial neoplasia (CIN), themethod comprising administering a vaccine according to any one ofembodiments 152-159 to a subject that suffers from CIN;

164) a method for treating cervical cancer, the method comprisingadministering a vaccine according to any one of embodiments 152-159 to asubject that suffers from cervical cancer;

165) a method for treating oropharyngeal cancer, the method comprisingadministering a vaccine according to any one of embodiments 152-159 to asubject that suffers from oropharyngeal cancer;

166) a method for treating penile intraepithelial neoplasia (PIN), themethod comprising administering a vaccine according to any one ofembodiments 152-159 to a subject that suffers from PIN;

167) a method for treating penile cancer, the method comprisingadministering a vaccine according to any one of embodiments 152-159 to asubject that suffers from penile cancer;

168) a method for treating vaginal intraepithelial neoplasia (VaIN), themethod comprising administering a vaccine according to any one ofembodiments 152-159 to a subject that suffers from VaIN;

169) a method for treating vaginal cancer, the method comprisingadministering a vaccine according to any one of embodiments 152-159 to asubject that suffers from vaginal cancer;

170) a method for treating anal intraepithelial neoplasia (AIN), themethod the method comprising administering a vaccine according to anyone of embodiments 152-159 to a subject that suffers from AIN;

171) a method for treating anal cancer, the method comprisingadministering a vaccine according to any one of embodiments 152-159 to asubject that suffers from anal cancer.

The practice of this disclosure will employ, unless otherwise indicated,conventional techniques of immunology, molecular biology, microbiology,cell biology, and recombinant DNA, which are within the skill of theart. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning. ALaboratory Manual, 2^(nd) edition, 1989; Current Protocols in MolecularBiology, F. M. Ausubel et al., eds., 1987; the series Methods inEnzymology (Academic Press, Inc.); PCR2: A Practical Approach, M. J.MacPherson, B. D. Hams, G. R. Taylor, eds., 1995; Antibodies: ALaboratory Manual, Harlow and Lane, eds., 1988.

The disclosure is further described by the following illustrativeexamples. The examples are not to limit the disclosure in any way.

EXAMPLES Example 1 Construction of a Designer Polypeptide ComprisingEssentially all HPV16 E6 and E7 CTL Epitopes

We designed a novel, non-tumorigenic polypeptide (and nucleic acidmolecule encoding such) that contains essentially all CTL epitopes ofHPV16 E6 and E7 proteins, and has a minimum number ofanticipated/predicted strong neo-epitopes (neo-epitopes meaning epitopesnot present in the wild type HPV16 E6 and E7 proteins) A polypeptide(also sometimes referred to as “E6E7SH” herein) comprises a sequence asprovided in SEQ ID NO: 1. A codon-optimized nucleic acid moleculeencoding this polypeptide is provided in SEQ ID NO:2.

The molecules are single molecules, which provides manufacturingadvantages over strategies where multiple molecules are used. Inaddition, the polypeptide of the disclosure comprises essentially allputative CTL epitopes that are present in wild-type E6 and E7 of HPV16,and at the same time have a minimum number of anticipated/predictedstrong neo-epitopes that could potentially be immunodominant and thusdivert the immune response from relevant wild-type CTL epitopes. Thusthe constructs of the disclosure are immunologically more favorable thanmolecules described by others that either lack possible CTL epitopesand/or that contain more or stronger neo-epitopes.

For instance, the construct of SEQ ID NO:1 contains only one neo-epitopewith a length of nine amino acids with a predicted binding affinity <50nM for the 20 most common HLA-A, 20 most common HLA-B and 20 most commonHLA-C alleles (HLA-A*01:01, HLA-A*02:01, HLA-A*02:03, HLA-A*02:06,HLA-A*02:07, HLA-A*03:01, HLA-A*11:01, HLA-A*23:01, HLA-A*24:02,HLA-A*26:01, HLA-A*29:02, HLA-A*30:01, HLA-A*30:02, HLA-A*31:01,HLA-A*32:01, HLA-A*33:01, HLA-A*33:03, HLA-A*34:01, HLA-A*68:01,HLA-A*68:02, HLA-B*07:02, HLA-B*07:04, HLA-B*08:01, HLA-B*13:01,HLA-B*15:01, HLA-B*18:01, HLA-B*35:01, HLA-B*37:01, HLA-B*39:01,HLA-B*40:01, HLA-B*40:02, HLA-B*40:06, HLA-B*44:02, HLA-B*44:03,HL-B*46:01, HLA-B*48:01, HLA-B*51:01, HLA-B*52:01, HLA-B*53:01,HLA-B*58:01, HLA-C*07:02, HLA-C*04:01, HLA-C*03:04, HLA-C*01:02,HLA-C*07:01, HLA-C*06:02, HLA-C*03:03, HLA-C*08:01, HLA-C*15:02,HLA-C*12:02, HLA-C*02:02, HLA-C*05:01, HLA-C*14:02, HLA-C*03:02,HLA-C*16:01, HLA-C*08:02, HLA-C*12:03, HLA-C*04:03, HLA-C*17:01,HLA-C*14:03), as determined using the ANN (Lundegaard et al., 2008,Nucl. Acids Res. 36:W509-12) and SMM method (Peters et al., 2003,Bioinformatics 19:1765-72) for HLA-A and HLA-B and the NetMHCpan method(Hoof et al., 2009, Immunogenetics 61:1-13) for HLA-C of the predictiontool for “Peptide binding to MHC class I molecules” at the IEDB website(at tools.immuneepitope.org/analyze/html/mhc_binding.html, version2009-09-01B).

As a non-limiting example, using the SMM prediction tool at the IEDBwebsite, the shuffled E6 and E7 sequences as described by Oosterhuis etal., 2011, Int. J. Cancer 129:397-406, and Öhischläger et al., 2006,Vaccine 24:2880-93 contain each nine potential strong uniqueneo-epitopes (ANN or SMM IC50<50 nM) for the 20 most HLA-A and -B, inthe core part. This even excludes the appendices used in that approach(in which appendices will further contribute to additional neo-epitopes,and may miss out on more native MHC II epitopes due to the limitedlength of the “overlap”). Indeed, a reportedly improved moleculecontaining a variant with shuffled E6 and E7 proteins that was describedin WO 2013/083287, contains 22 unique neo-epitopes with a length of nineamino acids with a predicted IC50<50 nM (ANN, SMM or NetMHCPan) for the20 most common HLA-A, 20 most common HLA-B and 20 most common HLA-Calleles.

Hence, the designer molecules hereof clearly are favorable in havingmuch lower number of predicted neo-epitopes compared to other publishedapproaches where E6 and E7 where shuffled to remove functionality.

Nucleic acid molecule encoding the thus designed HPV16 E6E7SH molecule(i.e., a polypeptide comprising SEQ ID NO:1) was synthesized, thepolynucleotide comprising SEQ ID NO:2, and flanked by a HindIII site anda Kozak sequence on the 5′ end and an XbaI site on the 3′ site (customsynthesis and standard molecular cloning at Invitrogen Lifetechnologies, Germany).

The synthesized fragments were cloned using HindIII and XbaI into astandard expression vector, pCDNA2004.Neo, harboring both a bacterialresistance marker (Ampicillin) and a mammalian resistance marker(Neomycin), to obtain plasmid vectors encoding a molecule of thedisclosure, e.g., for (transient) transfection based experiments.

These molecules could be used as such, but also as the basis for furthermolecules that contain additional features. As non-limiting examples,some further variants were prepared as described below.

The HPV16 E6E7SH fusion protein sequence can be combined with sequencesof other HPV16 early proteins to target individuals with persistentinfection and to broaden the immune repertoire in an immunizedindividual. Immune responses against E2 have been suggested to play animportant role in the clearance of HPV16 infections (de Jong et al.,2002, Cancer Res. 62:472-479). Fusion of E2 to E6E7SH will give avaccine component that harbors antigens against the stages ofHPV-related cancer from persistent infection to invasive cancer orrecurrent/refractory disease after LEEP surgery. Therefore, as anon-limiting example of such embodiments, we prepared a sequence codingfor a fusion protein of E6E7SH with E2 at its N-terminus. In the E2sequence modifications can be made to abrogate DNA binding activity thatmight affect gene expression in cells expressing the fusion protein. Wemutated Glycine at position 293, Lysine at position 299 and Cysteine atposition 300 of the wt HPV16 E2 protein into respectively Valine,Methionine and Arginine. Each of these mutations on its own alreadycompletely abrogates the binding of E2 to DNA sequences that harbor E2binding domains (Prakash et al., 1992, Genes Dev. 6:105-16).

The resulting polypeptide is referred to as HPV16 E2E6E7SH and comprisesSEQ ID NO:3. A codon-optimized sequence encoding this polypeptide wasprepared and is provided in SEQ ID NO:4.

We also constructed a variant wherein the same E2 mutant protein wasfused to the C-terminus of the HPV16 E6E7SH fusion polypeptide, givingrise to a polypeptide referred to as HPV16 E6E7E2SH, which comprises SEQID NO:5. The sequence encoding this construct is provided as SEQ IDNO:6.

For control purposes, we also constructed sequences encoding apolypeptide that contains the wild-type sequences for full-length HPV16E6 and E7 as a fusion protein (E6 from aa 1 to 158 directly fused to E7from aa 1 to 98, named herein E6E7 wt).

We also tested the effect of adding leader sequences to the polypeptide.As a non-limiting example, a sequence encoding an IgE leader sequence(see, e.g., U.S. Pat. No. 6,733,994) [the sequence of the leader peptideis provided in SEQ ID NO:7] was fused at the N-terminus of some of theconstructs, e.g., in the E6E7 wt construct, which rendered LSE6E7 wt,and in the E2E6E7SH construct, which rendered LSE2E6E7SH. The effectthereof was significantly (p<0.05) enhanced immunogenicity in comparisonto the same antigen without the LS sequence as measured by E7-tetrameranalysis in immunized mice (as can, for instance, be seen in FIGS.9A-9C).

The sequences that encode the E6E7SH polypeptides hereof, with orwithout E2, can, for instance, be expressed from DNA constructs, fromRNA or from viral vectors. FIG. 1 demonstrates expression in HEK-293Tcells upon transient transfection with DNA vectors expressing thetransgenes as described above. After transfection, cells were harvestedand cell extracts were analyzed by SDS-PAGE and western blotting with anantibody against HPV16 E7. This experiment demonstrates expression ofthe expected fusion proteins of appropriate size upon transfection ofthe expression vectors.

Adenoviral vectors can be used to express the E6E7, either with orwithout E2, and with or without additional sequences to augment theimmunogenicity of the encoded fusion protein.

The genes, coding for HPV16 E6E7 wt control or HPV designer sequencesdescribed above were gene optimized for human expression andsynthesized, by GENEART®. A Kozak sequence (5′ GCCACC 3′) was includeddirectly in front of the ATG start codon, and two stop codons (5′ TGATAA 3′) were added at the end of the respective coding sequence. Thegenes were inserted in the pAdApt35BSU plasmid and in the pAdApt26plasmid (Havenga et al., 2006, J. Gen. Virol. 87:2135-43) via HindIIIand XbaI sites.

All adenoviruses were generated in PER.C6® cells by single homologousrecombination and produced as previously described (for rAd35: Havengaet al., 2006, J. Gen. Virol. 87:2135-43; for rAd26: Abbink et al., 2007,J. Virol. 81:4654-63). PER.C6® cells (Fallaux et al., 1998, Hum. GeneTher. 9:1909-17) were maintained in Dulbecco's modified Eagle's medium(DMEM) with 10% fetal bovine serum (FBS), supplemented with 10 mM MgCl2.

Briefly, PER.C6® cells were transfected with Ad vector plasmids, usingLipofectamine according to the instructions provided by the manufacturer(Life Technologies). Cells were harvested one day after full cytopathiceffect (CPE) was reached, freeze-thawed, centrifuged for 5 min at 3,000rpm, and stored at −20° C. The viruses were plaque purified andamplified in PER.C6® cells cultured in a single well of a multiwell24-tissue culture plate. Further amplification was carried out inPER.C6® cells cultured in a T25 tissue culture flask and subsequently ina T175 tissue culture flask. Of the crude lysate prepared from the cellsobtained after the T175 flask, 3 to 5 ml was used to inoculate 24×T1000five-layer tissue culture flasks containing 70% confluent layers ofPER.C6® cells. The virus was purified using a two-step CsCl purificationmethod. Finally, the virus was stored in aliquots at −85° C.

Ad35.HPV16-E6E7 wt, and Ad35.HPV16-E6E7SH are recombinant adenovirusserotype 35 (Ad35) vectors comprising the codon-optimized nucleotidesequences for the expression of, respectively, a fusion protein of thewild type HPV16 E6 and E7 proteins (E6E7wt), and a designer fusionprotein variant as described above (E6E7SH, having the amino acidsequence provided in SEQ ID NO:1). The combined E6 and E7 sequences wereplaced under the control of a CMV promoter in the E1 region of the E1,E3 deleted adenovirus genome. Ad26.HPV16-E6E7wt, and Ad26.HPV16-E6E7SHare the equivalent vectors based on recombinant adenovirus serotype 26.

Similarly, Ad26 and Ad35-based recombinant adenoviral vectors wereproduced that encode the HPV16 E2E6E7SH (SEQ ID NO:3) variant. Likewise,Ad26 and Ad35 encoding the HPV16 E6E7E2SH (SEQ ID NO:5) variant wereproduced. Also, an Ad35 vector encoding the E2E6E7SH fusion protein withan IgE leader sequence at the N-terminus was produced, namedAd35.HPV16-LSE2E6E7SH. Also a control adenovirus with the E6E7 wt fusedto the IgE leader sequence at the N-terminus was produced.

The recombinant adenoviruses were produced on PER.C6® cells and purifiedby centrifugation on cesium chloride gradients.

Further examples of constructs of the disclosure that were coupled torepressor systems are provided in a later example below.

Example 2 Lack of Transforming Activity of the Designer Constructs

Wild-type HPV16 E6 and E7 proteins have tumorigenic potential, which isapparent as transforming activity in certain assays, such as colonyformation in a soft-agar assay (Massimi and Banks, 2005, Methods Mol.Med. 119:381-395). The E6E7SH polypeptide as described in Example 1comprises the fragments of the E6 and E7 proteins in a re-orderedfashion. This is expected to remove the tumorigenic potential, as can bemeasured, for instance, by a significantly reduced transforming activityas compared to either of wt E6 and E7 proteins in such assays.

Others reported that gene-shuffled variants of HPV16 E6 and E7 haveindeed lost their oncogenic potential (Öhlschläger et al., 2006, Vaccine24:2880-93; Henken et al., 2012, Vaccine 30:4259-66), demonstrating thatgene shuffling destroys the wild-type functions of E6 and E7 proteins.

To assess the loss of tumorigenic properties, we assessed the ability ofour E6E7SH constructs to confer the ability to grow in soft agar uponNIH 3T3 cells (as described by, e.g., Massimi and Banks, 2005, MethodsMol. Med. 119:381-395). Transfection of NIH3T3 cells with a plasmidexpressing the wild type HPV16 E7 resulted consistently in colonyformation. In these assays, expression of wild type HPV16 E6 alone didnot cause colony formation above background. This is in line withpublished observations that E7 wt is much more efficient than E6 wt inthis assay (Sedman et al., 1991, J. Virol. 65:4860-66). Transfectionwith our E6E7SH construct did not lead to growth of colonies of cells insoft agar (FIGS. 2A-2C) in four independent experiments, demonstratingthat nucleic acids encoding a polypeptide of the disclosure, E6E7SH,have lost the transforming capacity that is associated with E7.

The tumorigenic potential of E6 and E7 is associated with their abilityto reduce the levels of the cellular proteins p53 and pRb respectively.p53 and pRb degradation assays were performed to demonstrate thatnucleic acid molecule encoding a polypeptide of the disclosure, E6E7SH,construct does not have the biological activity associated with thewild-type E6 and E7 at the molecular level. In short, HPV16 E6 wt andour E6E7SH construct were expressed in NCI-H1299 cells that lackendogenous p53 for the p53 degradation assay. For the pRb degradationassay HPV16 E7 wt and the E6E7SH construct were expressed in pRb nullSaos-2 cells. As can be seen in FIGS. 3A-3D, co-expression of p53 withE6 wt, but not with E6E7SH, leads to reduced p53 levels (FIGS. 3A and3B). Likewise, FIGS. 3C and 3D show that co-expression of pRb with E7wt, but not with E6E7SH, leads to reduced pRB levels. These datademonstrate that nucleic acid molecule encoding a polypeptide of thedisclosure has no ability to form colonies in soft agar and does notcontain main biological activities of the wild-type E6 and E7polypeptides, namely the inactivation of p53 and pRb respectively.

To further demonstrate the safety of nucleic acid constructs encodingpolypeptide of the disclosure, we made use of primary human foreskinkeratinocytes that are the natural target cells for HPV mediatedtransformation. Immortalization of primary human keratinocytes requiresthe action of both E6 and E7 wild-type (Munger et al., 1989, J. Virol.63:4417-21). This assay is probably the physiologically most relevant invitro assay to demonstrate the safety of our constructs (Massimi andBanks, 2005, Methods Mol. Med. 119:381-395). Cells transduced withlentiviruses expressing wild type E6 and E7 from HPV16 (E6E7 wt) induceimmortalization in primary keratinocytes as indicated by the extensionof their lifespan as compared to non-transduced control cells (FIG. 4)and activation of hTERT, the catalytic subunit of telomerase (data notshown). Expression of the polypeptide of the disclosure (E6E7SH) is notable to extend the lifespan compared to GFP-transduced or non-transducedkeratinocytes. A similar result was obtained in two additionalindependent donors (data not shown). Taken together these datademonstrate that our constructs have lost the ability to induceimmortalization in primary human keratinocytes that are considered ahighly physiological model.

Another construct wherein fragments of HPV16 E6 and E7 were recombinedin another order was also incapable of immortalization of primary humanforeskin keratinocytes. However, an expanded life span up toapproximately 120-150 days was observed for that construct. Thisindicates some unpredictability in this field, and demonstrates thesuperiority of the designer molecules as described herein in thissafety-related aspect.

All together the experiments in this example provide strong evidence ofthe lack of transforming activity of nucleic acids encoding polypeptidesas described herein, and thus a strongly improved safety over HPV16 E6and E7 wt constructs.

Example 3 Immune Responses to the E6E7SH Designer Constructs

We have prepared DNA vectors and adenoviral vectors, as described inExample 1.

We used the CB6F1 mouse strain for measuring immune responses, based oninitial experiments where mice where immunized with DNA plasmidsencoding wild type E2, or E6 or E7, and immunization with HPV16 E2, E6and E7 antigens induced a broader cellular immune response in CB6F1 thanin C57BL/6 mice or Balb/c mice. In a separate experiment mice wereimmunized with DNA vectors encoding molecules of the disclosure andcellular immune responses were measured. HPV16 E7-specific immuneresponses could be measured in mice immunized with DNA plasmidsexpressing E6E7SH (FIGS. 5A and 5B).

The following data shown in this example are from mouse experiments thatwere carried out with adenoviral vectors.

To evaluate the vaccine induced immunogenicity, CB6F1 mice wereimmunized with adenovectors (Ad35) expressing E6E7 wt, LSE6E7 wt, E6E7SHor adenovectors not encoding a transgene (Empty). Two doses were testedfor administration to the mice: 5*109 viral particles (vp) and 1*10¹⁰vp. Two and eight weeks after immunization the mice were sacrificed andisolated splenocytes were stimulated overnight with an HPV16 E7 15merpeptide pool. E7-specific responses at two weeks and at eight weeks wereanalyzed by IFNγ ELISPOT. The data are presented in FIGS. 6A-6C.

This shows that immunization of mice with Ad35.HPV16-E6E7SH inducesE7-specific immune responses as measured by ELISPOT analysis. Inaddition, the results in FIGS. 6A-6C demonstrate the possibility toenhance the immune response against an adenoviral expressed transgene byadding an N-terminal leader sequence to the transgene.

Next the effect of adding E2 to the E6E7SH polypeptide with respect toimmunogenicity was tested. The Ad35 vectors encoded polypeptides thathad E2 either fused to the N-terminus (E2E6E7SH) or to the C-terminus(E6E7E2SH). CB6F1 mice were immunized with a dose of 1×10¹⁰ vp. FIGS. 7Aand 7B (E7-tetramer staining) and FIG. 8C (IFNγ ELISPOT) show the immuneresponses against E7, which for the designer constructs including E2tends to be higher in comparison to the construct without E2, althoughthe differences were not statistically significant. The response againstE2 was higher for adenoviral vectors encoding only E2 compared to theresponse for adenoviral vectors that had E2 fused to the E6E7SH designerpolypeptide (FIG. 8B), with differences being significant for both E2 vsE2E6E7SH and E2 vs E6E7E2SH (p-value: <0.05).

It is concluded that the designer constructs that further include E2 canstill provide an immune response against E7, and in addition alsoprovide an immune response against E2, thus increasing the breadth ofthe immune response over the constructs that do not include E2.

Addition of a leader sequence was shown to result in higher E7-specificresponses when fused to the N-terminus of the fusion protein of wildtype E6 and E7 (FIG. 6C). Similarly, the effect of the leader sequenceon immunogenicity of the E2E6E7SH fusion protein was determined.Therefore, Ad35 vectors encoding the designer polypeptide, with orwithout N-terminal E2 and an Ad35 vector encoding LSE2E6E7SH were usedfor immunization of mice and blood samples were taken at two-weekintervals to measure E7-specific immune responses (FIGS. 9A-9C). Asshown in FIGS. 7A, 7B, and 8A-8C, the presence of E2 at either N- orC-terminally fused to E6E7SH tended to increase the immune responses.Addition of the IgE leader sequence further increased the E7-specificresponse (FIG. 9B). Over time sustained immune responses were observedfor all three adenoviral vectors that encoded designer molecules asdescribed herein, and the highest response after the immunizationcorresponded with the highest responses over the duration of theexperiment.

It is concluded that the responses that are induced by the designerconstruct that further includes N-terminal E2 can be increased byaddition of specific sequences, e.g., the IgE leader sequence, thattarget the encoded protein to specific cellular compartments.

The cellular immune response against the peptide of the disclosure canbe induced with different types of adenoviral vectors. In the previousexperiment we used Ad35 vectors, while in the experiment of FIGS. 10Aand 10B, mice were immunized with an Ad26 adenoviral vector expressingE2E6E7SH. The data show that also immunization with an Ad26-basedvaccine induced E7-specific T-cells. In addition, the resultsdemonstrate that a second immunization with an Ad35 adenoviral vectorexpressing E2E6E7SH further boosted the cellular immune responses (FIGS.10A and 10B).

Example 4 Immunogenicity of Designer Constructs in Rhesus Macaques

To evaluate the ability of the adenoviral vectors expressing thedesigner sequence of the disclosure to induce immune responses innon-human primates, rhesus macaques were immunized by intramuscularinjection with adenovectors (Ad26) expressing E2E6E7SH or adenovectorsnot encoding a transgene (Empty), with a dose of 1*10¹¹ vp. Eight weeksafter the immunization the immune responses were boosted by immunizationwith Ad26 vectors expressing the same antigen. At week 16 the animalsreceived one more injection with the Ad35 vectors expressing the sameantigen. Blood samples were taken at several time points and isolatedwhite blood cells were stimulated overnight with a peptide poolscorresponding to HPV16 E2, E6 or E7. Specific responses were measured byIFNγ ELISPOT. The data are presented in FIGS. 11A and 11B. In additionat week 10 and week 18 post-prime immunization, the cellular immuneresponse specific to peptides spanning the novel junctions in thedisclosure was evaluated. The induction of IFNγ response was in allanimals below the limit of detection of <50 SFU per 1*10⁶ PBMC (data notshown).

The data show that immunization of non-human primates withAd26.HPV16-E2E6E7SH resulted in cellular immune responses against allthree HPV16 proteins that are present in the encoded transgene, but notagainst the novel junctions. Responses could be boosted by theadditional immunization with Ad26.HPV16-E2E6E7SH and additional boost atweek 16 with the corresponding Ad35 vector further increased the HPV16E2, E6 and E7-specific immune responses.

A late booster administration at week 72 with Ad26.HPV16-E2E6E7SH againresulted in an increase of the HPV16 cellular immune response, whichafter a few weeks was declined (not shown).

In a separate experiment (not shown), Rhesus macaques were immunized byintravaginal administration with a combination of two adenoviralvectors, one expressing HPV16 E6E7SH and the other the HPV16 L1 protein.Low but measurable cellular responses were measured in peripheralmononuclear blood cells against both E6 and E7. In these experiments,strong cellular immune responses against L1 were detected.

Example 5 Therapeutic Efficacy in a Mouse Tumor Model

The polypeptide of the disclosure is capable of inducing HPV16-specificcellular immune response in animals, which can exert a therapeuticeffect on cells expressing HPV16 E6 and/or E7. Therapeutic immunization,i.e., immunization after tumor growth has started, can be used todemonstrate efficacy of a therapeutic HPV vaccine candidate. Thetherapeutic effect of Ad26 and Ad35 vectors was tested in mice that wereinjected with TC-1 cells (mouse cells expressing HPV16 E6 and E7) (Linet al., 1996, Cancer Res. 56:21-6). TC-1 cells will form solid tumorwithin a few days to weeks after sub-cutaneous injection in mice.Without vaccine the tumors grew rapidly and reach a pre-determined sizeof 1000 mm³ within 30 days (FIGS. 12D and 12E). Upon reaching this sizethe mice are sacrificed for ethical reasons.

With a prime-boost immunization scheme with SLPs (used as a positivecontrol; Kenter et al., 2009, N. Engl. J. Med. 361:1838-47; Zwaveling etal., 2002, J. Immunol. 169:350-8) or adenoviral vectors expressingHPV16-E2E6E7SH, a marked decrease of the growth of TC-1 induced tumorswas observed (FIGS. 12B and 12C). Closer inspection of the first 30 daysafter the prime immunizations (FIGS. 12F and 12G) shows that theimmunization with the adenovectors expressing E2E6E7SH have asubstantially larger impact on tumor growth than immunization with theSLPs. The initial growth rate is much lower and in most cases the tumorsshrunk. In 3 out of 11 mice immunized with the adenoviral vectors, thetumors were completely eradicated, which is reflected in the survivalplot (FIG. 12H).

In conclusion, immunization with adenoviral vectors expressing apolypeptide of the disclosure significantly reduced tumor growth orcompletely eradicated established tumors in a well-established challengemodel for HPV16-induced cancer.

Example 6 Employment of Repressor Systems to Improve the Productivityand Genetic Stability of Adenoviral Vectors Expressing HPV-DerivedAntigens

It has previously been reported that transgenes inserted into adenovirusvectors under the control of powerful constitutively active promoterscan, depending on the properties of the transgene product, negativelyimpact vector production (Yoshida and Yamada, 1997, Biochem. Biophys.Res. Commun. 230:426-30; Rubinchik et al., 2000, Gene Ther. 7:875-85;Matthews et al., 1999, J. Gen. Virol. 80:345-53; Edholm et al., 2001, J.Virol. 75:9579-84; Gall et al., 2007, Mol. Biotechnol. 35:263-73).Examples of transgene-dependent vector productivity issues includeinefficient vector rescue and growth, low final vector yields, and, insevere cases, rapid outgrowth of viral mutants with defective transgenecassettes. To solve these issues, multiple studies explored thepossibility to silence vector transgene expression during vectorreplication in producer cells (Matthews et al., 1999, J. Gen. Virol.80:345-53; Edholm et al., 2001, J Virol. 75:9579-84; Gall et al., 2007,Mol. Biotechnol. 35:263-73; Cottingham et al., 2012, Biotechnol. Bioeng.109:719-28; Gilbert et al., 2014, J. Virol. Methods 208:177-88). In thisregard, different repression systems have previously been implemented inthe context of Ad vectors and have indeed shown to improve vectorproductivity and genetic stability for vectors encoding different typesof (inhibitory) transgenes.

It was observed that some of the adenovirus vectors described herein, aswell as some other adenoviral vectors encoding certain HPV antigenvariants, displayed some of the transgene-dependent vector productivityissues described above and, therefore, could possibly be furtherimproved in that respect. We, therefore, sought to investigate whetherusage of systems to repress vector transgene expression can improveproduction characteristics of Ad vectors expressing HPV-derived antigensas those described herein. For this purpose, we implemented two existingrepressor-operator systems, i.e., TetR/TetO (Yao and Eriksson, 1999,Hum. Gene Ther. 10:419-22, EP0990041B1) and CymR/CuO (Mullick et al.,2006, BMC Biotechnol. 6:43), into our adenovirus vector platform. Boththe TetR/TetO and the CymR/CuO system have previously been used byothers to improve adenovirus vector productivity through vectortransgene silencing during vector replication (Gall et al., 2007, Mol.Biotechnol. 35:263-73; Cottingham et al., 2012, Biotechnol. Bioeng.109:719-28; Gilbert et al., 2014, J. Virol. Methods 208:177-88).Implementation of these two systems involved the generation ofadenoviral vectors expressing genes of interest under the control ofeither a TetO or a CuO sequence-containing CMV promoter. Furthermore,the implementation entailed the generation of cell lines stablyexpressing the respective cognate repressors proteins (i.e., TetR orCymR).

Several E1-deleted, Ad26- and Ad35-based vectors were generated in whichsequences encoding heterologous polypeptides were operably linked to aCMV promoter containing either TetO or CuO operator sequences. First,certain TetO- or CuO-containing sequences (SEQ ID NO: 11 and SEQ IDNO:12, respectively) were inserted near the transcription start site(TSS) of the CMV promoter (SEQ ID NO:13) of pAdapt26 and pAdapt35.Bsuplasmids (Abbink et al., 2007, J. Virol. 81:4654-63; Havenga et al.,2006, J. Gen. Virol. 87:2135-43). The operator-containing sequences wereinserted at precisely the same positions of the CMV promoter aspreviously described for the two systems (Yao and Eriksson, 1999, HumanGene Ther. 10:419-22; EP0990041B1; Mullick et al., 2006, BMC Biotechnol.6:43; EP1385946B1). Specifically, relative to the TSS (as originallyassigned; Stenberg et al., 1984, J Virol. 49:190-9), the TetO- andCuO-containing sequences were inserted directly downstream of positions−20 and +7, respectively. In SEQ ID NO:13, these two positionscorrespond to positions 716 and 742, respectively. The resultingoperator-containing CMV promoters are termed, respectively, CMVTetO andCMVCuO. Next, different transgenes were inserted downstream of the(modified) CMV promoters of the resulting constructs using HindIII andXbaI restriction sites. These transgenes included genes encoding afusion protein of green fluorescent protein and luciferase (GFP-Luc),LSE2E6E7SH from the present disclosure, and another polypeptide withsome similarity to LSE2E6E7SH (a construct referred to in this exampleas “HPVAg”). HPVAg comprises the same leader sequence as present inLSE2E6E7SH, as well as E2, E6, and E7 sequences of HPV16. Using methodsas described herein, the resulting modified pAdapt26 and pAdapt35.Bsuplasmids were used for the generation of adenoviral vectors expressingthe above mentioned reporter and HPV transgenes under the control ofeither the CMVTetO or the CMVCuO promoter.

Cell lines expressing either TetR or CymR were generated by stabletransfection of PER.C6® cells using, respectively, plasmid pcDNA™6/TR(LifeTechnologies, V1025-20) and a derivative of pcDNA™6/TR in which theTetR-coding sequence (SEQ ID NO:14, which encodes polypeptide SEQ IDNO:15) is replaced by a codon-optimized CymR-coding sequence (SEQ IDNO:16, which encodes polypeptide SEQ ID NO:17). Stable cell linegeneration was performed largely as described by the supplier ofpcDNA™6/TR using a transient transfection-based assay to screen for cellclones capable of repressing expression of CMVTetO- or CMVCuO-drivengenes. The resulting PER.C6/TetR and PER.C6/CymR cell lines wereanalyzed for their ability to repress transgene expression during vectorreplication in these cells. Experiments conducted with vectorsexpressing GFP-Luc under the control of operator-containingCMV-promoters showed at least a ten-fold reduction of luciferase geneexpression throughout the complete virus replication cycle in the celllines expressing the repressor corresponding to the respective operatorsequences (data not shown). This confirmed that the PER.C6/TetR andPER.C6/CymR cell lines were capable of repressing vector transgeneexpression in the context of replicating adenovirus vectors.

The effect of TetR- and CymR-mediated repression of adenovectortransgene expression on vector yields was investigated for Ad35-basedvectors expressing HPVAg (FIG. 13A). To this end, PER.C6®, PER.C6/TetR,and PER.C6/CymR cell lines, seeded at 3*10⁵ cells per well in 24-wellplate wells, were subjected to quadruplicate infections—at 1000 virusparticles per cell and for a duration of three hours—by vectorsexpressing HPVAg from either CMVTetO or CMVCuO promoters. As controls,parallel infections were performed with corresponding vectors expressingGFP-Luc instead of HPVAg. Four days after infection, crude viral lysateswere prepared by subjecting the contents of the wells (i.e., infectedcells and medium) to two freeze-thaw cycles. Adenovector titers weresubsequently determined by an Ad35 hexon sequence-specific quantitativePCR-based protocol that uses a purified Ad35 vector with known virusparticle titer as a standard. The results show that both the TetO- andthe CuO-containing HPVAg-encoding Ad35 vectors, compared to the controlvectors expressing GFP-Luc, display decreased vector yields on normalPER.C6® cells. By contrast, when produced on cells expressing theircognate repressors (i.e., TetR and CymR, respectively), these samevectors gave yields as high as those obtained with the control vectors.These data indicate that repression of transgene expression duringvector production in producer cells can be beneficial for theproductivity of Ad35 vectors carrying HPVAg as a transgene.

The effect that repression of adenovector transgene expression may haveon vector yields was also investigated for vectors derived fromadenovirus serotype 26 (Ad26) (FIG. 13B). In an assay performedessentially as described above for the Ad35 vectors, Ad26 vectorscarrying CMVTetO promoter-controlled transgenes encoding either GFP-Luc,HPVAg, or LSE2E6E7SH were used to infect PER.C6® and PER.C6/TetR cellsat 1500 virus particles per cell. Three days later the infections wereharvested and virus particle titers determined by an Ad26 hexonsequence-specific quantitative PCR-based method. The results show thaton PER.C6® cells the yields for the vectors encoding HPVAg andLSE2E6E7SH are lower than obtained with the control vector encodingGFP-Luc. In contrast, on PER.C6/TetR cells, both these vectors showedtiters that are as high as that obtained for the control vector.Together with the results above (for Ad35 vectors), these data indicatethat repression of transgene expression during adenovector productionincreases the yields of vectors expressing HPVAg and LSE2E6E7SH.

We have observed major issues regarding the genetic stability of anadenovirus vector that carried a CMV promoter-driven transgene forHPVAg. For example, it was observed that after several passaging roundsof this vector on PER.C6® the majority of the vector populationconsisted of a mutant vector that carried a large deletion in the HPVAgcoding sequence (data not shown).

We reasoned that employment of a transgene expression repression system,such as one of the two described above, could prevent genetic stabilityissues associated with transgenes, such as HPVAg that are inhibitory tovector growth. To test this, an Ad35-based vector with CMVCuOpromoter-driven HPVAg expression was assessed for transgene cassettestability upon growth of the vector on either PER.C6® or PER.C6/CymRcells (FIGS. 14A, and 14B). In brief, vector DNA was transfected intothe two different cell lines and resultant viral plaques were allowed togrow under an agarose layer. From each of the two transfections, fiveviral plaques were isolated and separately passaged further on the samecell line (i.e., as used for the transfection), for ten consecutiveviral passages. Transgene integrity was assessed by PCR amplification ofthe transgene cassette at viral passage number ten (VPN10), and thesubsequent analysis of resultant PCR products by gel electrophoresis andSanger sequencing. In addition, at VPN7, the passaged viral clones wereassessed for their ability to express HPVAg. This was done by using thepassaged viral isolates to infect A549 cells at 1000 virus particles percell, lysing the cells at 48 hours post-infection, and subsequentlyanalyzing the expression of HPVAg by western blotting using a monoclonalantibody directed against HPV16 E7 (Santa-Cruz Biotechnology). Theresults of the gel electrophoresis and sequencing analyses showed thatall five viral isolates that had been passaged on PER.C6® each carriedeither small frameshifting deletions or premature stop mutations withinthe transgene cassette. By contrast, such deletions or mutations couldnot be detected in any of the vector isolates that had been passaged onthe cell line expressing CymR (PER.C6/CymR). In agreement with thesedata, all PER.C6/CymR-propagated vector isolates were able to expressHPVAg, while all PER.C6®-grown vectors completely lost this ability,suggesting defective transgene cassettes for these vectors. Inconclusion, our data demonstrate that employment of a repressor system,as, for instance, the CymR/CuO system, to repress vector transgeneexpression during vector propagation is an effective means to preventsevere transgene cassette instability, such as that seen for vectorscarrying a transgene expressing HPVAg.

REFERENCES The Contents of Each of which are Incorporated Herein by thisReference

-   Abbink P, A. A. Lemckert, B. A. Ewald, D. M. Lynch, M. Denholtz, S.    Smits, L. Holterman, I. Damen, R. Vogels, A. R. Thorner, K. L.    O'Brien, A. Carville, K. G. Mansfield, J. Goudsmit, M. J. Havenga,    and D. H. Barouch (2007). Comparative seroprevalence and    immunogenicity of six rare serotype recombinant adenovirus vaccine    vectors from subgroups B and D. J. Virol. 81:4654-4663.-   Ausubel F. M. (1995). Short protocols in molecular biology: a    compendium of methods from Current Protocols in Molecular Biology.    Wiley, [Chichester].-   Cottingham M. G., F. Carroll, S. J. Morris, A. V. Turner, A. M.    Vaughan, M. C. Kapulu, S. Colloca, L. Siani, S. C. Gilbert,    and A. V. Hill (2012). Preventing spontaneous genetic rearrangements    in the transgene cassettes of adenovirus vectors. Biotechnol.    Bioeng. 109:719-728.-   Daayana S., E. Elkord, U. Winters, M. Pawlita, R. Roden, P. L.    Stern, and H. C. Kitchener (2010). Phase II trial of imiquimod and    HPV therapeutic vaccination in patients with vulval intraepithelial    neoplasia. Br. J. Cancer 102:1129-1136.-   de Jong A., S. H. van der Burg, K. M. Kwappenberg, J. M. van der    Hulst, K. L. Franken, A. Geluk, K. E. van Meijgaarden, J. W.    Drijfhout, G. Kenter, P. Vermeij, C. J. Melief, and R. Offringa    (2002). Frequent detection of human papillomavirus 16 E2-specific    T-helper immunity in healthy subjects. Cancer Res. 62:472-479.-   Edholm D., M. Molin, E. Bajak, and G. Akusjarvi (2001). Adenovirus    vector designed for expression of toxic proteins. J. Virol.    75:9579-9584.-   Evans R. K., D. K. Nawrocki, L. A. Isopi, D. M. Williams, D. R.    Casimiro, S. Chin, M. Chen, D. M. Zhu, J. W. Shiver, and D. B.    Volkin (2004). Development of stable liquid formulations for    adenovirus-based vaccines. J. Pharm. Sci. 93:2458-2475.-   Fallaux F. J., A. Bout, 1. van der Velde, D. J. van den    Wollenberg, K. M. Hehir, J. Keegan, C. Auger, S. J. Cramer, H. van    Ormondt, A. J. van der Eb, D. Valerio, and R. C. Hoeben (1998). New    helper cells and matched early region 1-deleted adenovirus vectors    prevent generation of replication-competent adenoviruses. Hum. Gene    Ther. 9:1909-1917.-   Frøkjær S., and L. Hovgaard (2000). Pharmaceutical formulation    development of peptides and proteins. Taylor and Francis, London.-   Gall J. G., A. Lizonova, D. EttyReddy, D. McVey, M. Zuber, I.    Kovesdi, B. Aughtman, C. R. King, and D. E. Brough (2007). Rescue    and production of vaccine and therapeutic adenovirus vectors    expressing inhibitory transgenes. Mol. Biotechnol. 35:263-273.-   Gao G. P., R. K. Engdahl, and J. M. Wilson (2000). A cell line for    high-yield production of E1-deleted adenovirus vectors without the    emergence of replication-competent virus. Hum. Gene Ther.    11:213-219.-   Gennaro A. R. (1990). Remington's Pharmaceutical Sciences. Mack-   Gilbert R., C. Guilbault, D. Gagnon, A. Bernier, L. Bourget, S. M.    Elahi, A. Kamen, and B. Massie (2014). Establishment and validation    of new complementing cells for production of E1-deleted adenovirus    vectors in serum-free suspension culture. J. Virol. Methods    208:177-188.-   Hamid O., and R. D. Carvajal (2013). Anti-programmed death-1 and    anti-programmed death-ligand 1 antibodies in cancer therapy. Expert    Opin. Biol. Ther. 13:847-861.-   Harlow E., and D. Lane (1988). Antibodies: A Laboratory Manual. Cold    Spring Harbor Laboratory, New York.-   Havenga M., R. Vogels, D. Zuijdgeest, K. Radosevic, S. Mueller, M.    Sieuwerts, F. Weichold, I. Damen, J. Kaspers, A. Lemckert, M. van    Meerendonk, R. van der Vlugt, L. Holterman, D. Hone, Y. Skeiky, R.    Mintardjo, G. Gillissen, D. Barouch, J. Sadoff, and J. Goudsmit    (2006). Novel replication-incompetent adenoviral B-group vectors:    high vector stability and yield in PER.C6® cells. J. Gen. Virol.    87:2135-2143.-   Henken F. E., K. Oosterhuis, P. Ohlschlager, L. Bosch, E.    Hooijberg, J. B. Haanen, and R. D. Steenbergen (2012). Preclinical    safety evaluation of DNA vaccines encoding modified HPV16 E6 and E7.    Vaccine 30:4259-4266.-   Hildesheim A., R. Herrero, S. Wacholder, A. C. Rodriguez, D.    Solomon, M. C. Bratti, J. T. Schiller, P. Gonzalez, G. Dubin, C.    Porras, S. E. Jimenez, and D. R. Lowy (2007). Effect of human    papillomavirus 16/18 L1 virus-like particle vaccine among young    women with preexisting infection: a randomized trial. JAMA    298:743-753.-   Hoganson D. K., J. C. Ma, L. Asato, M. Ong, M. A. Printz, B. G.    Huyghe, B. A. Sosnowshi, and M. J. D'Andrea (2002). Development of a    stable adenoviral vector formulation. Bioprocess J. 1:43-48.-   Hoof I., B. Peters, J. Sidney, L. E. Pedersen, A. Sette, O. Lund, S.    Buus, and M. Nielsen (2009). NetMHCpan, a method for MHC class I    binding prediction beyond humans. Immunogenetics 61:1-13.-   Horwitz M. S. (1996). “Adenoviruses” in B. N. Fields, D. M.    Knipe, J. D. Baines (eds.), Virology. Raven Press Ltd, New York.-   Kenter G. G., M. J. Welters, A. R. Valentijn, M. J. Lowik, D. M.    Berends-van der Meer, A. P. Vloon, F. Essahsah, L. M. Fathers, R.    Offringa, J. W. Drijfhout, A. R. Wafelman, J. Oostendorp, G. J.    Fleuren, S. H. van der Burg, and C. J. Melief (2009). Vaccination    against HPV-16 oncoproteins for vulvar intraepithelial neoplasia. N.    Engl. J. Med. 361:1838-1847.-   Kibbe A. H. (2000). Handbook of Pharmaceutical Excipients.    Pharmaceutical Press, London.-   Kovesdi I., and S. J. Hedley (2010). Adenoviral producer cells,    Viruses 2:1681-1703.-   Lin K. Y., F. G. Guarnieri, K. F. Staveley-O'Carroll, H. I.    Levitsky, J. T. August, D. M. Pardoll, and T. C. Wu (1996).    Treatment of established tumors with a novel vaccine that enhances    major histocompatibility class II presentation of tumor antigen.    Cancer Res. 56:21-26.-   Lundegaard C., K. Lamberth, M. Harndahl, S. Buus, O. Lund, and M.    Nielsen (2008). NetMHC-3.0: accurate web accessible predictions of    human, mouse and monkey MHC class I affinities for peptides of    length 8-11. Nucleic Acids Res. 36:W509-512.-   Massimi P., and L. Banks (2005). “Transformation Assays for HPV    Oncoproteins” in C. Davy, J. Doorbar (eds.), Human Papillomaviruses:    Methods and Protocols, Vol 119: Methods in Molecular Medicine.    Springer, Berlin, pp. 381-395.-   Matthews D. A., D. Cummings, C. Evelegh, F. L. Graham, and L. Prevec    (1999). Development and use of a 293 cell line expressing lac    repressor for the rescue of recombinant adenoviruses expressing high    levels of rabies virus glycoprotein. J. Gen. Virol. 80 (Pt    2):345-353.-   McPherson M. J., B. D. Hames, G. R. Taylor (1995). PCR 2: A    Practical Approach. IRL Press at Oxford University Press, Oxford.-   Melman I., G. Coukos, and G. Dranoff (2011). Cancer immunotherapy    comes of age. Nature 480:480-489.-   Mullick A., Y. Xu, R. Warren, M. Koutroumanis, C. Guilbault, S.    Broussau, F. Malenfant, L. Bourget, L. Lamoureux, R. Lo, A. W.    Caron, A. Pilotte, and B. Massie (2006). The cumate gene-switch: a    system for regulated expression in mammalian cells. BMC Biotechnol.    6:43.-   Munger K., W. C. Phelps, V. Bubb, P. M. Howley, and R. Schlegel    (1989). The E6 and E7 genes of the human papillomavirus type 16    together are necessary and sufficient for transformation of primary    human keratinocytes. J. Virol. 63:4417-4421.-   Ogun S. A., L. Dumon-Seignovert, J. B. Marchand, A. A. Holder,    and F. Hill (2008). The oligomerization domain of C4-binding protein    (C4 bp) acts as an adjuvant, and the fusion protein comprised of the    19-kilodalton merozoite surface protein 1 fused with the murine C4    bp domain protects mice against malaria. Infect. Immun.    76:3817-3823.-   Oosterhuis K., E. Aleyd, K. Vrijland, T. N. Schumacher, and J. B.    Haanen (2012a). Rational Design of DNA Vaccines for the Induction of    Human Papillomavirus Type 16 E6- and E7-Specific Cytotoxic T-Cell    Responses. Hum. Gene Ther. 23:1301-1312.-   Oosterhuis K., P. Ohlschlager, J. H. van den Berg, M. Toebes, R.    Gomez, T. N. Schumacher, and J. B. Haanen (2011). Preclinical    development of highly effective and safe DNA vaccines directed    against HPV16 E6 and E7. Int. J. Cancer 129:397-406.-   Oosterhuis K., J. H. van den Berg, T. N. Schumacher, and J. B.    Haanen (2012b). DNA vaccines and intradermal vaccination by DNA    tattooing. Curr. Top Microbiol. Immunol. 351:221-250.-   Peters B., W. Tong, J. Sidney, A. Sette, and Z. Weng (2003).    Examining the independent binding assumption for binding of peptide    epitopes to MHC-I molecules. Bioinformatics 19:1765-1772.-   Prakash S. S., S. R. Grossman, R. B. Pepinsky, L. A. Laimins,    and E. J. Androphy (1992). Amino acids necessary for DNA contact and    dimerization imply novel motifs in the papillomavirus E2    trans-activator. Genes Dev. 6:105-116.-   Rubinchik S., R. Ding, A. J. Qiu, F. Zhang, and J. Dong (2000).    Adenoviral vector which delivers FasL-GFP fusion protein regulated    by the tet-inducible expression system. Gene Ther. 7:875-885.-   Sambrook JFEFMIT (1989). Molecular cloning: A Laboratory Manual.    Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.-   Sedman S. A., M. S. Barbosa, W. C. Vass, N. L. Hubbert, J. A.    Haas, D. R. Lowy, and J. T. Schiller (1991). The full-length E6    protein of human papillomavirus type 16 has transforming and    trans-activating activities and cooperates with E7 to immortalize    keratinocytes in culture. J. Virol. 65:4860-4866.-   Shenk T. (1996). “Adenoviridae and their Replication” in B. N.    Fields, D. M. Knipe, and J. D. Baines (eds.), Virology. Raven Press    Ltd, New York.-   Smahel M., P. Sima, V. Ludvikova, and V. Vonka (2001). Modified    HPV16 E7 Genes as DNA Vaccine against E7-Containing Oncogenic Cells.    Virology 281:231-238.-   van der Burg S. H., and C. J. Melief (2011). Therapeutic vaccination    against human papilloma virus induced malignancies. Curr. Opin.    Immunol. 23:252-257.-   Watson J. D. (1992). Recombinant DNA. Scientific American Books, New    York.-   Wieking B. G., D. W. Vermeer, W. C. Spanos, K. M. Lee, P.    Vermeer, W. T. Lee, Y. Xu, E. S. Gabitzsch, S. Balcaitis, J. P.    Balint, Jr., F. R. Jones, and J. H. Lee (2012). A non-oncogenic    HPV16 E6/E7 vaccine enhances treatment of HPV expressing tumors.    Cancer Gene Ther. 19:667-674.-   Yan J., D. K. Reichenbach, N. Corbitt, D. A. Hokey, M. P.    Ramanathan, K. A. McKinney, D. B. Weiner, and D. Sewell (2009).    Induction of antitumor immunity in vivo following delivery of a    novel HPV-16 DNA vaccine encoding an E6/E7 fusion antigen. Vaccine    27:431-440.-   Yao F., and E. Eriksson (1999). A novel tetracycline-inducible viral    replication switch. Hum. Gene Ther. 10:419-427.-   Yoshida Y., and H. Hamada (1997). Adenovirus-mediated inducible gene    expression through tetracycline-controllable transactivator with    nuclear localization signal. Biochem. Biophys. Res. Commun.    230:426-430.-   Yugawa T., and T. Kiyono (2009). Molecular mechanisms of cervical    carcinogenesis by high-risk human papillomaviruses: novel functions    of E6 and E7 oncoproteins. Rev. Med. Virol. 19:97-113.-   Zwaveling S., S. C. Ferreira Mota, J. Nouta, M. Johnson, G. B.    Lipford, R. Offringa, S. H. van der Burg, and C. J. Melief (2002).    Established human papillomavirus type 16-expressing tumors are    effectively eradicated following vaccination with long peptides. J.    Immunol. 169:350-358.

TABLE I sequencesSEQ ID NO: 1 (HPV16-E6E7SH, amino acid sequence of HPV16 E6/E7 designer polypeptide)MHQKRTAMFQ DPQERPRKLP QLCTELQTTI HDIILECVYC KQQLEDEIDGPAGQAEPDRA HYNIVTFCCK CDSTLRLCVQ STHVDIRTLE DLLMGTLGIVCPICSQKPGT TLEQQYNKPL CDLLIRCINC QKPLCPEEKQ RHLDKKQRFHNIRGRWTGRC MSCCRSSRTR RETQMHGDTP TLHEYMLDLQ PETTDLYCYEQLNDSSEEED EIDGPAGQAE PDRAHYNIVT FCCQLCTELQ TTIHDIILECVYCKQQLLRR EVYDFAFRDL CIVYRDGNPY AVCDKCLKFY SKISEYRHYCYSLYGTTLEQ QYNKPLCDLL IRCINCQKSEQ ID NO: 2 (HPV16-E6E7SH, nucleotide sequence encoding amino acid sequence of HPV16E6/E7 designer polypeptide)ATGCACCAGA AACGGACCGC CATGTTCCAG GACCCCCAGG AACGGCCCAGAAAGCTGCCC CAGCTGTGCA CCGAGCTGCA GACCACCATC CACGACATCATCCTGGAATG CGTGTACTGC AAGCAGCAGC TGGAAGATGA GATCGACGGCCCTGCTGGCC AGGCCGAACC CGACAGAGCC CACTACAATA TCGTGACCTTCTGCTGCAAG TGCGACAGCA CCCTGCGGCT GTGCGTGCAG AGCACCCACGTGGACATCCG GACCCTGGAA GATCTGCTGA TGGGCACCCT GGGCATCGTGTGCCCCATCT GCAGCCAGAA GCCCGGCACC ACCCTGGAAC AGCAGTACAACAAGCCCCTG TGCGACCTGC TGATCCGGTG CATCAACTGC CAGAAACCCCTGTGCCCCGA GGAAAAGCAG CGGCACCTGG ACAAGAAGCA GCGGTTCCACAACATCCGGG GCAGATGGAC AGGCAGATGC ATGAGCTGCT GCAGAAGCAGCCGGACCAGA CGGGAAACCC AGATGCACGG CGACACCCCC ACCCTGCACGAGTACATGCT GGACCTGCAG CCCGAGACAA CCGACCTGTA CTGCTACGAGCAGCTGAACG ACAGCAGCGA GGAAGAGGAC GAGATTGACG GACCCGCTGGACAGGCCGAG CCTGACCGGG CTCACTATAA CATCGTGACA TTTTGCTGTCAGCTCTGTAC TGAACTCCAG ACAACAATTC ACGATATTAT TCTCGAATGTGTGTATTGTA AACAGCAGCT CCTGCGGAGA GAGGTGTACG ACTTCGCCTTCCGGGACCTC TGCATCGTGT ATCGGGACGG CAACCCCTAC GCCGTGTGCGACAAGTGCCT GAAGTTCTAC AGCAAGATCA GCGAGTACCG GCACTACTGCTACAGCCTGT ACGGAACAAC ACTCGAACAG CAGTATAACA AACCACTCTGTGATCTGCTG ATTCGCTGTA TCAATTGTCA GAAGTGATAASEQ ID NO: 3 (HPV16 E2E6E7SH, amino acid sequence of HPV16 E2/E6/E7 designerpolypeptide)METLCQRLNVCQDKILTHYENDSTDLRDHIDYWKHMRLECAIYYKAREMGFKHINHQVVPTLAVSKNKALQAIELQLTLETIYNSQYSNEKWTLQDVSLEVYLTAPTGCIKKHGYTVEVQFDGDICNTMHYTNWTHIYICEEASVTVVEGQVDYYGLYYVHEGIRTYFVQFKDDAEKYSKNKVWEVHAGGQVILCPTSVFSSNEVSSPEIIRQHLANHPAATHTKAVALGTEETQTTIQRPRSEPDTGNPCHTTKLLHRDSVDSAPILTAFNSSHKGRINCNSNTTPIVHLKVDANTLMRLRYRFKKHCTLYTAVSSTWHWTGHNVKHKSAIVTLTYDSEWQRDQFLSQVKIPKTTTVSTGFMSIMHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPGTTLEQQYNKPLCDLLIRCINCQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQMHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKSEQ ID NO: 4 (HPV16 E2E6E7SH, nucleotide sequence encoding HPV16 E2/E6/E7 designerpolypeptideATGGAAACCCTGTGCCAGCGGCTGAACGTGTGCCAGGACAAGATCCTGACCCACTACGAGAACGACAGCACCGACCTGCGGGACCACATCGACTACTGGAAGCACATGCGGCTGGAATGCGCCATCTACTACAAGGCCAGAGAGATGGGCTTCAAGCACATCAACCACCAGGTGGTGCCCACCCTGGCCGTGTCCAAGAACAAGGCCCTGCAGGCCATCGAGCTGCAGCTGACCCTGGAAACCATCTACAACAGCCAGTACAGCAACGAGAAGTGGACCCTGCAGGACGTGTCCCTGGAAGTGTACCTGACCGCTCCCACCGGCTGCATCAAGAAACACGGCTACACCGTGGAAGTGCAGTTCGACGGCGACATCTGCAACACCATGCACTACACCAACTGGACCCACATCTACATCTGCGAAGAGGCCAGCGTGACCGTGGTGGAAGGCCAGGTGGACTACTACGGCCTGTACTACGTGCACGAGGGCATCCGGACCTACTTCGTGCAGTTCAAGGACGACGCCGAGAAGTACAGCAAGAACAAAGTGTGGGAGGTGCACGCTGGCGGCCAGGTCATCCTGTGCCCCACCAGCGTGTTCAGCAGCAACGAGGTGTCCAGCCCCGAGATCATCCGGCAGCACCTGGCCAATCACCCTGCCGCCACCCACACAAAGGCCGTGGCCCTGGGCACCGAGGAAACCCAGACCACCATCCAGCGGCCCAGAAGCGAGCCCGACACCGGCAATCCCTGCCACACCACCAAGCTGCTGCACCGGGACAGCGTGGACAGCGCCCCTATCCTGACCGCCTTCAACAGCAGCCACAAGGGCCGGATCAACTGCAACAGCAACACCACCCCCATCGTGCACCTGAAGGTGGACGCCAACACCCTGATGCGGCTGCGGTACAGATTCAAGAAGCACTGCACCCTGTACACCGCCGTGTCCTCCACCTGGCACTGGACCGGCCACAACGTGAAGCACAAGAGCGCCATCGTGACCCTGACCTACGACAGCGAGTGGCAGCGGGACCAGTTCCTGAGCCAGGTCAAAATCCCCAAGACCATCACCGTGTCCACCGGCTTCATGAGCATCATGCACCAGAAACGGACCGCCATGTTCCAGGACCCCCAGGAACGGCCCAGAAAGCTGCCCCAGCTGTGCACCGAGCTGCAGACCACCATCCACGACATCATCCTGGAATGCGTGTACTGCAAGCAGCAGCTGGAAGATGAGATCGACGGCCCTGCTGGCCAGGCCGAACCCGACAGAGCCCACTACAATATCGTGACCTTCTGCTGCAAGTGCGACAGCACCCTGCGGCTGTGCGTGCAGAGCACCCACGTGGACATCCGGACCCTGGAAGATCTGCTGATGGGCACCCTGGGCATCGTGTGCCCCATCTGCAGCCAGAAGCCCGGCACCACCCTGGAACAGCAGTACAACAAGCCCCTGTGCGACCTGCTGATCCGGTGCATCAACTGCCAGAAACCCCTGTGCCCCGAGGAAAAGCAGCGGCACCTGGACAAGAAGCAGCGGTTCCACAACATCCGGGGCAGATGGACAGGCAGATGCATGAGCTGCTGCAGAAGCAGCCGGACCAGACGGGAAACCCAGATGCACGGCGACACCCCCACCCTGCACGAGTACATGCTGGACCTGCAGCCCGAGACAACCGACCTGTACTGCTACGAGCAGCTGAACGACAGCAGCGAGGAAGAGGACGAGATTGACGGACCCGCTGGACAGGCCGAGCCTGACCGGGCTCACTATAACATCGTGACATTTTGCTGTCAGCTCTGTACTGAACTCCAGACAACAATTCACGATATTATTCTCGAATGTGTGTATTGTAAACAGCAGCTCCTGCGGAGAGAGGTGTACGACTTCGCCTTCCGGGACCTCTGCATCGTGTATCGGGACGGCAACCCCTACGCCGTGTGCGACAAGTGCCTGAAGTTCTACAGCAAGATCAGCGAGTACCGGCACTACTGCTACAGCCTGTACGGAACAACACTCGAACAGCAGTATAACAAACCACTCTGTGATCTGCTGATTCGCTGTATCAATTGTCAGAAGTGATAASEQ ID NO: 5 (HPV16 E6E7E2SH, encoding HPV16 E6/E7/E2 designer polypeptideMHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKPGTTLEQQYNKPLCDLLIRCINCQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQMHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKMETLCQRLNVCQDKILTHYENDSTDLRDHIDYWKHMRLECAIYYKAREMGFKHINHQVVPTLAVSKNKALQATELQLTLETIYNSQYSNEKWTLQDVSLEVYLTAPTGCIKKHGYTVEVQFDGDICNTMHYTNWTHIYICEEASVTVVEGQVDYYGLYYVHEGIRTYFVQFKDDAEKYSKNKVWEVHAGGQVILCPTSVFSSNEVSSPEIIRQHLANHPAATHTKAVALGTEETQTTIQRPRSEPDTGNPCHTTKLLHRDSVDSAPILTAFNSSHKGRINCNSNTTPIVHLKVDANTLMRLRYRFKKHCTLYTAVSSTWHWTGHNVKHKSAIVTLTYDSEWQRDQFLSQVKIPKTITVSTGFMSISEQ ID NO: 6 (HPV16 E6E7E2SH, nucleotide sequence encoding HPV16 E6/E7/E2 designerpolypeptideATGCACCAGAAACGGACCGCCATGTTCCAGGACCCCCAGGAACGGCCCAGAAAGCTGCCCCAGCTGTGCACCGAGCTGCAGACCACCATCCACGACATCATCCTGGAATGCGTGTACTGCAAGCAGCAGCTGGAAGATGAGATCGACGGCCCTGCTGGCCAGGCCGAACCCGACAGAGCCCACTACAATATCGTGACCTTCTGCTGCAAGTGCGACAGCACCCTGCGGCTGTGCGTGCAGAGCACCCACGTGGACATCCGGACCCTGGAAGATCTGCTGATGGGCACCCTGGGCATCGTGTGCCCCATCTGCAGCCAGAAGCCCGGCACCACCCTGGAACAGCAGTACAACAAGCCCCTGTGCGACCTGCTGATCCGGTGCATCAACTGCCAGAAACCCCTGTGCCCCGAGGAAAAGCAGCGGCACCTGGACAAGAAGCAGCGGTTCCACAACATCCGGGGCAGATGGACAGGCAGATGCATGAGCTGCTGCAGAAGCAGCCGGACCAGACGGGAAACCCAGATGCACGGCGACACCCCCACCCTGCACGAGTACATGCTGGACCTGCAGCCCGAGACAACCGACCTGTACTGCTACGAGCAGCTGAACGACAGCAGCGAGGAAGAGGACGAGATTGACGGACCCGCTGGACAGGCCGAGCCTGACCGGGCTCACTATAACATCGTGACATTTTGCTGTCAGCTCTGTACTGAACTCCAGACAACAATTCACGATATTATTCTCGAATGTGTGTATTGTAAACAGCAGCTCCTGCGGAGAGAGGTGTACGACTTCGCCTTCCGGGACCTCTGCATCGTGTATCGGGACGGCAACCCCTACGCCGTGTGCGACAAGTGCCTGAAGTTCTACAGCAAGATCAGCGAGTACCGGCACTACTGCTACAGCCTGTACGGAACAACACTCGAACAGCAGTATAACAAACCACTCTGTGATCTGCTGATTCGCTGTATCAATTGTCAGAAGATGGAAACCCTGTGCCAGCGGCTGAACGTGTGCCAGGACAAGATCCTGACCCACTACGAGAACGACAGCACCGACCTGCGGGACCACATCGACTACTGGAAGCACATGCGGCTGGAATGCGCCATCTACTACAAGGCCAGAGAGATGGGCTTCAAGCACATCAACCACCAGGTGGTGCCCACCCTGGCCGTGTCCAAGAACAAGGCCCTGCAGGCCATCGAGCTGCAGCTGACCCTGGAAACCATCTACAACAGCCAGTACAGCAACGAGAAGTGGACCCTGCAGGACGTGTCCCTGGAAGTGTACCTGACCGCTCCCACCGGCTGCATCAAGAAACACGGCTACACCGTGGAAGTGCAGTTCGACGGCGACATCTGCAACACCATGCACTACACCAACTGGACCCACATCTACATCTGCGAAGAGGCCAGCGTGACCGTGGTGGAAGGCCAGGTGGACTACTACGGCCTGTACTACGTGCACGAGGGCATCCGGACCTACTTCGTGCAGTTCAAGGACGACGCCGAGAAGTACAGCAAGAACAAAGTGTGGGAGGTGCACGCTGGCGGCCAGGTCATCCTGTGCCCCACCAGCGTGTTCAGCAGCAACGAGGTGTCCAGCCCCGAGATCATCCGGCAGCACCTGGCCAATCACCCTGCCGCCACCCACACAAAGGCCGTGGCCCTGGGCACCGAGGAAACCCAGACCACCATCCAGCGGCCCAGAAGCGAGCCCGACACCGGCAATCCCTGCCACACCACCAAGCTGCTGCACCGGGACAGCGTGGACAGCGCCCCTATCCTGACCGCCITCAACAGCAGCCACAAGGGCCGGATCAACTGCAACAGCAACACCACCCCCATCGTGCACCTGAAGGTGGACGCCAACACCCTGATGCGGCTGCGGTACAGATTCAAGAAGCACTGCACCCTGTACACCGCCGTGTCCTCCACCTGGCACTGGACCGGCCACAACGTGAAGCACAAGAGCGCCATCGTGACCCTGACCTACGACAGCGAGTGGCAGCGGGACCAGTTCCTGAGCCAGGTCAAAATCCCCAAGACCATCACCGTGTCCACCGGCTTCATGAGCATCTGATAASEQ ID NO: 7 (IgE leader peptide amino acid sequence) MDWTWILFLVAAATRVHSSEQ ID NO: 8 (nucleotide sequence encoding IgE leader peptide)ATGGACTGGACCTGGATCCTGTTCCTGGTGGCTGCCGCAACCCGGGTGCACAGCSEQ ID NO: 9 (aa HAVT20 leader peptide amino acid sequence)MACPGFLWALVISTCLEFSMASEQ ID NO: 10 (nucleotide sequence encoding HAVT20 leader peptide)ATGGCCTGCCCCGGCTTTCTGTGGGCCCTGGTCATCAGCACCTGTCTGGAATTCAGCATGGCCSEQ ID NO: 11 (2xTetO-containing sequence)GAGCTCTCCCTATCAGTGATAGAGATCTCCCTATCAGTGATAGAGATCGTCGACSEQ ID NO: 12 (CuO-containing sequence) AACAAACAGACAATCTGGTCTGTTTGTASEQ ID NO: 13 (CMV promoter present in pAdApt26 and pAdApt35 plasmids)TCAATATTGGCCATTAGCCATATTATTCATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATTGCATACGTTGTATCCATATCATAATATGTACATTTATATTGGCTCATGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGASEQ ID NO: 14 (TetR, nucleotide sequence encoding amino acid sequence of TetR polypeptideexpressed by pcDNA ™ 6/TR)ATGTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTAATGAGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTAGAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTTAGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAAGCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTCATCGCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAAACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCATTATATGCACTCAGCGCTGTGGGGCATTTTACTTTAGGTTGCGTATTGGAAGATCAAGAGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACTGATAGTATGCCGCCATTATTACGACAAGCTATCGAATTATTTGATCACCAAGGTGCAGAGCCAGCCTTCTTATTCGGCCTTGAATTGATCATATGCGGATTAGAAAAACAACTTAAATGTGAAAGTGGGTCCGCGTACAGCGGATCCCGGGAATTCAGATCTTATTAASEQ ID NO: 15 (TetR, amino acid sequence of TetR polypeptide expressed by pcDNA ™6/TR) MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSGSREFRSYSEQ ID NO: 16 (CymR, nucleotide sequence encoding amino acid sequence of CymRpolypeptide)ATGTCTCCCAAACGACGGACTCAAGCGGAAAGGGCAATGGAAACTCAGGGTAAGCTGATTGCCGCGGCTCTGGGAGTGCTGCGAGAGAAAGGGTATGCCGGGTTTCGCATAGCCGACGTTCCTGGAGCTGCAGGCGTAAGCAGAGGAGCCCAATCTCATCACTTTCCGACCAAGCTGGAGCTTTTGCTGGCTACCTTCGAATGGCTGTACGAGCAGATCACGGAAAGGAGTCGTGCTAGGCTGGCCAAGCTGAAACCCGAGGATGATGTCATTCAGCAGATGCTGGACGATGCAGCCGAGTTCTTCCTGGACGACGACTTCAGCATCAGTCTCGACCTCATCGTAGCCGCAGATCGCGATCCAGCTTTGCGCGAGGGCATACAGAGAACAGTCGAGCGGAATCGGTTTGTGGTGGAGGACATGTGGCTTGGTGTTCTGGTGAGCAGAGGCCTCTCACGGGATGATGCCGAGGACATCCTGTGGCTGATCTTTAACTCCGTCAGAGGGTTGGCAGTGAGGTCCCTTTGGCAGAAGGACAAAGAACGGTTTGAACGTGTGCGAAACTCAACACTCGAGATTGCTAGGGAACGCTACGCCAAGTTCAAGAGATGASEQ ID NO: 17 (CymR, amino acid sequence of CymR polypeptide)MSPKRRTQAERAMETQGKLIAAALGVLREKGYAGFRIADVPGAAGVSRGAQSHHEPTKLELLLATFEWLYEQITERSRARLAKLKPEDDVIQQMLDDAAEFFLDDDFSISLDLIVAADRDPALREGIQRTVERNRFVVEDMWLGVLVSRGLSRDDAEDILWLIFNSVRGLAVRSLWQKDKERFERVRNSTLEIARERYAKFKR SEQ ID NO: 18 (HPV16 E6, aa41-65) KQQLLRREVYDFAFRDLCIVYRDGNSEQ ID NO: 19 (HPV16 E7 aa 43-77) GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR

What is claimed is:
 1. A nucleic acid molecule encoding a polypeptidecomprising SEQ ID NO:1.
 2. A nucleic acid molecule according to claim 1,wherein the encoded polypeptide further comprises a leader sequence. 3.The nucleic acid molecule of claim 1, wherein the encoded polypeptidefurther comprises at least one epitope of a human papillomavirus (HPV)E2 protein.
 4. A nucleic acid molecule according to claim 3, wherein theencoded polypeptide comprises HPV16 E2 protein that has a deletion ormutation in its DNA binding domain.
 5. A nucleic acid molecule accordingto claim 4, wherein the encoded polypeptide comprises SEQ ID NO:3 or SEQID NO:5.
 6. The nucleic acid molecule of claim 1, wherein the nucleicacid sequence is codon-optimized.
 7. The nucleic acid molecule of claim1, comprising SEQ ID NO:2.
 8. The nucleic acid molecule of claim 1,comprising SEQ ID NO:4 or SEQ ID NO:6.
 9. A vector comprising thenucleic acid molecule of claim 1, wherein a sequence encoding thepolypeptide is operably linked to a promoter.
 10. A vector according toclaim 9, wherein the vector is a recombinant adenovirus.
 11. The vectorof claim 9, wherein the promoter is operably coupled to a repressoroperator sequence, to which a repressor protein can bind in order torepress expression of the promoter in the presence of said repressorprotein.
 12. A vaccine composition comprising: the vector of claim 9,and a pharmaceutically acceptable excipient.
 13. A method of inducing animmune response against HPV in a subject, the method comprising:administering to the subject the vaccine composition according to claim12.
 14. A The method according to claim 13, wherein the vaccinecomposition is administered to the subject more than once.
 15. A methodfor treating a subject having persistent HPV infection, vulvarintraepithelial neoplasia (VIN), cervical intraepithelial neoplasia(CIN), vaginal intraepithelial neoplasia (VaIN), anal intraepithelialneoplasia (AIN), cervical cancer (such as cervical squamous cellcarcinoma (SCC), oropharyngeal cancer, penile cancer, vaginal cancer oranal cancer, the method comprising: administering to the subject thevaccine according to claim
 12. 16. A polypeptide comprising SEQ ID NO:1.17. The polypeptide according to claim 16, comprising SEQ ID NO:3 or SEQID NO:5.
 18. A recombinant vector comprising: a nucleic acid moleculeencoding a polypeptide comprising SEQ ID NO:1, operably linked to apromoter.
 19. The recombinant vector of claim 18, wherein the encodedpolypeptide further comprises at least one epitope of a humanpapillomavirus (HPV) E2 protein.
 20. The recombinant vector of claim 19,wherein the encoded polypeptide comprises HPV16 E2 protein that has adeletion or mutation in its DNA binding domain.
 21. The recombinantvector of claim 20, wherein the encoded polypeptide comprises SEQ IDNO:3 or SEQ ID NO:5.